Human Choroid Research Articles

Elevation of Granulocyte Colony Stimulating Factor in Human AMD Donor RPE-Choroid.

Choroidal inflammation, complement deposition, and accumulation of C-reactive protein (CRP) are involved in age-related macular degeneration (AMD) pathology. The pro-inflammatory signals that regulate immune cell recruitment in the choroid of patients with AMD remain to be determined. We performed cytokine profiling of human AMD and age-matched control donor tissue to identify inflammatory molecules upregulated in AMD tissue. Protein was isolated from 25 AMD and 21 control donor RPE/choroid macular punches. Total protein was quantified, and 50 µg assayed for expression of 40 cytokines using an inflammation array. We validated the elevated expression of granulocyte colony stimulating factor (G-CSF) protein by ELISA in a second cohort of 22 control and 26 AMD donors. To identify an AMD associated stressor responsible for upregulating G-CSF we assayed for changes in G-CSF protein secretion in RPE/choroid organ cultures treated with the monomeric (m)CRP, an inflammatory protein elevated in AMD. Using a multiplex array, we identified elevated G-CSF protein in the choroid of AMD donors compared to age-matched non-AMD controls. Differential expression of G-CSF was confirmed via ELISA in an independent cohort of samples (P = 0.01). The mCRP, which is deposited in AMD choroids, increased G-CSF protein secretion in RPE/choroid organ cultures. Single nuclei RNA sequencing identified choroidal endothelial cells and fibroblasts as the primary cell types responsible for increased G-CSF secretion in response to mCRP. The G-CSF receptor is expressed primarily by choroidal macrophages and dendritic cells and anti-G-CSFR colocalizes with anti-CD45 and anti-CD68 in human donor choroid tissue. Elevated G-CSF expression in AMD donor tissue as a result of increased levels of mCRP may be involved in immune cell recruitment in AMD contributing to inflammatory stress in the choroid.

Single-nucleus RNA-seq dissection of choroid plexus tumor cell heterogeneity.

The genomic, genetic and cellular events regulating the onset, growth and survival of rare, choroid plexus neoplasms remain poorly understood. Here, we examine the heterogeneity of human choroid plexus tumors by single-nucleus transcriptome analysis of 23,906 cells from four disease-free choroid plexus and eleven choroid plexus tumors. The resulting expression atlas profiles cellular and transcriptional diversity, copy number alterations, and cell-cell interaction networks in normal and cancerous choroid plexus. In choroid plexus tumor epithelial cells, we observe transcriptional changes that correlate with genome-wide methylation profiles. We further characterize tumor type-specific stromal microenvironments that include altered macrophage and mesenchymal cell states, as well as changes in extracellular matrix components. This first single-cell dataset resource from such scarce samples should be valuable for divising therapies against these little-studied neoplasms.

Tissue-Specific Immune Transcriptional Signatures in the Bordering Tissues of the Mouse Retina and Brain.

Bordering the central nervous system (CNS) parenchyma are the choroid (underlying the retina) and the leptomeninges (the inner layers of the meninges enveloping the brain). Although near the neural parenchyma, the choroid and leptomeninges are external to the immune privileged environment of the retina and brain and thus are distinct immune compartments. This study aimed to characterize the transcriptomic signatures of immune cells within the choroid and leptomeninges bordering the healthy adult mouse CNS. Eyes and brains were obtained from 7-week-old C57Bl/6J mice. Choroid and leptomeninges were processed for isolation of CD45+ immune cells and single cell RNA-sequencing. Additionally, single cell RNA-sequencing was performed on immune cells isolated from choroid obtained from human donor eye tissue. Immunostaining and confocal microscopy of wholemount tissue were used to validate selected immune cell populations in situ. A total of 3606 cells were sequenced from mouse tissues, including 2125 CD45+ cells from choroid and 1481 CD45+ cells from leptomeninges. Clustering and differential gene expression analysis revealed heterogeneous subtypes of monocytes/macrophages, dendritic cells, T cells, and B cells. Whereas some clusters were common to both choroid and leptomeninges, others exhibited tissue-specific gene expression profiles and potential functional specializations. Analysis of 6501 CD45+ cells sequenced from human choroid identified similar immune cell populations to mouse choroid. This study provides a detailed characterization of the molecular signatures of immune cells within the vascular connective tissues bordering the healthy retina and brain, and their potential roles in immune protection.

Non-endothelial expression of endomucin in the mouse and human choroid

Endomucin (EMCN) is a 261 amino acid transmembrane glycoprotein that is highly expressed by venous and capillary endothelial cells where it plays a role in VEGF-mediated angiogenesis and regulation of immune cell recruitment. However, it is better known as a histological marker, where it has become widespread due to the commercial availability of high-quality antibodies that work under a wide range of conditions and in many tissues. The specificity of EMCN staining has been well-validated in retinal vessels, but while it has been used extensively as a marker in other tissues of the eye, including the choroid, the pattern of expression has not been described in detail. Here, in addition to endothelial expression in the choriocapillaris and deeper vascular layers, we characterize a population of EMCN-positive perivascular cells in the mouse choroid that did not co-localize with cells expressing other endothelial markers such as PECAM1 or PODXL. To confirm that these cells represented a new population of EMCN-expressing stromal cells, we then performed single cell RNA sequencing in choroids from adult wild-type mice. Analysis of this new dataset confirmed that, in addition to endothelial cells, Emcn mRNA expression was present in choroidal pericytes and a subset of fibroblasts, but not vascular smooth muscle cells. Besides Emcn, no known endothelial gene expression was detected in these cell populations, confirming that they did not represent endothelial-stromal doublets, a common technical artifact in single cell RNA seq datasets. Instead, choroidal Emcn-expressing fibroblasts exhibited high levels of chemokine and interferon signaling genes, while Emcn-negative fibroblasts were enriched in genes encoding extracellular matrix proteins. Emcn expressing fibroblasts were also detected in published datasets from mouse brain and human choroid, suggesting that stromal Emcn expression was not unique to the choroid and was evolutionarily conserved. Together, these findings highlight unique fibroblast and pericyte populations in the choroid and provide new context for the role of EMCN in the eye.

Melanopsin in the human and chicken choroid

The choroid embedded in between retina and sclera is essential for retinal photoreceptor nourishment, but is also a source of growth factors in the process of emmetropization that converts retinal visual signals into scleral growth signals. Still, the exact control mechanisms behind those functions are enigmatic while circadian rhythms are involved. These rhythms are attributed to daylight influences that are melanopsin (OPN4) driven. Recently, OPN4-mRNA has been detected in the choroid, and while its origin is unknown we here seek to identify the underlying structures using morphological methods.Human and chicken choroids were prepared for single- and double-immunohistochemistry of OPN4, vasoactive intestinal peptide (VIP), substance P (SP), CD68, and α-smooth muscle actin (ASMA). For documentation, light-, fluorescence-, and confocal laser scanning microscopy was applied. Retinal controls proved the reliability of the OPN4 antibody in both species.In humans, OPN4 immunoreactivity (OPN4-IR) was detected in nerve fibers of the choroid and adjacent ciliary nerve fibers. OPN4+ choroidal nerve fibers lacked VIP, but were co-localized with SP. OPN4-immunoreactivity was further detected in VIP+/SP + intrinsic choroidal neurons, in a hitherto unclassified CD68-negative choroidal cell population thus not representing macrophages, as well as in a subset of choroidal melanocytes. In chicken, choroidal nerve fibers were OPN4+, and further OPN4-IR was detected in clustered suprachoroidal structures that were not co-localized with ASMA and therefore do not represent non-vascular smooth-muscle cells. In the choroidal stroma, numerous cells displayed OPN4-IR, the majority of which was VIP-, while a few of those co-localized with VIP and were therefore classified as avian intrinsic choroidal neurons. OPN4-immunoreactivity was absent in choroidal blood vessels of both species.In summary, OPN4-IR was detected in both species in nerve fibers and cells, some of which could be identified (ICN, melanocytes in human), while others could not be classified yet. Nevertheless, the OPN4+ structures described here might be involved in developmental, light-, thermally-driven or nociceptive mechanisms, as known from other systems, but with respect to choroidal control this needs to be proven in upcoming studies.

THE ORGANIZATION OF THE BASEMENT MEMBRANES IN THE CHOROID PLEXUS VILLI OF THE HUMAN BRAIN

THE ORGANIZATION OF THE BASEMENT MEMBRANES IN THE Choroid PLEXUS VILLI OF THE HUMAN BRAIN Authors O.V. Kirik 1*, O.S. Alekseeva 1,2, I.P. Grigorev 1, E.A. Fedorova 1, A.A. Beketova 1, D.E. Korzhevsky 1 Affiliation 1 Institute of Experimental Medicine, Saint Petersburg, Russian Federation 2 Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences, Saint Petersburg, Russian Federation Abstract BACKGROUND: The choroid plexus of the brain is the main component of the blood-cerebrospinal fluid barrier, separating these two biological fluids. Basement membranes of the choroid plexus have a special role in the implementation of barrier functions: they underlie the choroidal epithelium and capillary endothelium, and serve as an additional filter for substances penetrating from the blood into the cerebrospinal fluid. AIM: The purpose of this work was to study the organization of basement membranes in the villi of the human telencephalon choroid plexus using immunohistochemical detection of type IV collagen. METHODS: The study was performed on archival material from the choroid plexus of the human brain (age 29-50 years, n=10) using the immunohistochemical method for detecting type IV collagen. RESULTS: An immunohistochemical reaction using antibodies to type IV collagen showed the distribution of this protein in the subepithelial area and in the stroma of the choroid plexus villi. All immunopositive structures had clear contours; there was no reaction in the cell cytoplasm or a nonspecific background. Contacts of subepithelial and subcapillary basement membranes labeled with antibodies to type IV collagen were not detected. CONCLUSION: The study showed that the basement membranes of the villi of the choroid plexus of the human brain in the subepithelial and perivascular areas have a different organization. In this case, the union of the subepithelial and perivascular components containing type IV collagen, as a rule, does not occur.

Highly restrictive and directional penetration of the blood cerebral spinal fluid barrier by JCPyV.

The human polyomavirus JCPyV is an opportunistic pathogen that infects greater than 60% of the world's population. The virus establishes a persistent and asymptomatic infection in the urogenital system but can cause a fatal demyelinating disease in immunosuppressed or immunomodulated patients following invasion of the CNS. The mechanisms responsible for JCPyV invasion into CNS tissues are not known but direct invasion from the blood to the cerebral spinal fluid via the choroid plexus has been hypothesized. To study the potential of the choroid plexus as a site of neuroinvasion, we used an adult human choroid plexus epithelial cell line to model the blood-cerebrospinal fluid (B-CSF) barrier in a transwell system. We found that these cells formed a highly restrictive barrier to virus penetration either as free virus or as virus associated with extracellular vesicles (EVJC+). The restriction was not absolute and small amounts of virus or EVJC+ penetrated and were able to establish foci of infection in primary astrocytes. Disruption of the barrier with capsaicin did not increase virus or EVJC+ penetration leading us to hypothesize that virus and EVJC+ were highly cell-associated and crossed the barrier by an active process. An inhibitor of macropinocytosis increased virus penetration from the basolateral (blood side) to the apical side (CSF side). In contrast, inhibitors of clathrin and raft dependent transcytosis reduced virus transport from the basolateral to the apical side of the barrier. None of the drugs inhibited apical to basolateral transport suggesting directionality. Pretreatment with cyclosporin A, an inhibitor of P-gp, MRP2 and BCRP multidrug resistance transporters, restored viral penetration in cells treated with raft and clathrin dependent transcytosis inhibitors. Because choroid plexus epithelial cells are known to be susceptible to JCPyV infection both in vitro and in vivo we also examined the release of infectious virus from the barrier. We found that virus was preferentially released from the cells into the apical (CSF) chamber. These data show clearly that there are two mechanisms of penetration, direct transcytosis which is capable of seeding the CSF with small amounts of virus, and infection followed by directional release of infectious virions into the CSF compartment.

Response of the human choroid to short-term changes in eyelid and periocular temperature

ABSTRACT Clinical relevance Choroidal thickness measurement is gaining popularity in clinical practice and research as an early indicator of myopia progression. Understanding the influence of temperature on choroidal thickness changes will improve the reliability of the measures. Background It has been suggested that environmental temperature may affect choroidal thickness and blood flow, with potential implications for ocular disease and refractive development. This study investigates the effect of changes in eyelid/ocular adnexa temperature on choroidal thickness. Methods In a paired-eye study, 20 young, healthy subjects received a warm stimulus (heat pack) over one closed eye and simultaneously a cold stimulus (ice pack) over the other for 10 min. Eyelid temperatures were monitored with thermal probes, and optical coherence tomography scans of the retina and choroid were taken before and after heating and cooling, and then every 5 min during a 15-min recovery period. Retinal and choroidal thicknesses were measured across the macular region (6 mm), including the subfoveal (1 mm), parafoveal (1–3 mm), and perifoveal (3–5 mm) regions, and compared between the cooled and warmed eyes. Results When the thermal stimuli were applied, eyelid surface temperatures changed predictably and remained significantly different (by approximately 10–15°C) between the eyes after 2 min (p < .001). Relative to the warmed eye, macular choroidal thickness in the cooled eye increased significantly after 10 min of treatment (p = .004). This choroidal thickening response occurred in the subfoveal, parafoveal, and perifoveal regions (all p < .05). Upon removal of the thermal stimuli, choroidal thickness rapidly returned to the baseline and was no longer different between the cooled and warmed eye (p = .641). Conclusion Cooling the anterior eye by application of a cold stimulus directly onto the closed eyelid caused a small but significant increase in choroidal thickness relative to warming the anterior eye, demonstrating that the choroid can modulate its thickness rapidly and transiently in response to local temperature changes.

FABP4 Is an Indispensable Factor for Regulating Cellular Metabolic Functions of the Human Retinal Choroid.

The purpose of the current study was to elucidate the physiological roles of intraocularly present fatty acid-binding protein 4 (FABP4). Using four representative intraocular tissue-derived cell types, including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cells, the intraocular origins of FABP4 were determined by qPCR analysis, and the intracellular functions of FABP4 were investigated by seahorse cellular metabolic measurements and RNA sequencing analysis using a specific inhibitor for FABP4, BMS309403. Among these four different cell types, FABP4 was exclusively expressed in HOCF cells. In HOCF cells, both mitochondrial and glycolytic functions were significantly decreased to trace levels by BMS309403 in a dose-dependent manner. In the RNA sequencing analysis, 67 substantially up-regulated and 94 significantly down-regulated differentially expressed genes (DEGs) were identified in HOCF cells treated with BMS309403 and those not treated with BMS309403. The results of Gene Ontology enrichment analysis and ingenuity pathway analysis (IPA) revealed that the DEGs were most likely involved in G-alpha (i) signaling, cAMP-response element-binding protein (CREB) signaling in neurons, the S100 family signaling pathway, visual phototransduction and adrenergic receptor signaling. Furthermore, upstream analysis using IPA suggested that NKX2-1 (thyroid transcription factor1), HOXA10 (homeobox A10), GATA2 (gata2 protein), and CCAAT enhancer-binding protein A (CEBPA) were upstream regulators and that NKX homeobox-1 (NKX2-1), SFRP1 (Secreted frizzled-related protein 1) and TREM2 (triggering receptor expressed on myeloid cells 2) were causal network master regulators. The findings in this study suggest that intraocularly present FABP4 originates from the ocular choroid and may be a critical regulator for the cellular homeostasis of non-adipocyte HOCF cells.

Modeling Mild Traumatic Brain Injury In-Vitro Using PHuman Choroid Plexus Endothelial Cells

Over 2.5 million Traumatic Brain Injuries (TBIs) occur each year due to falls, motor vehicle accidents, assaults, and sports (Brain Trauma Foundation, 2023). Short- and long-term consequences of TBI can include headache, cognitive impairment, seizures, hypoxia, and ischemia. Human TBI studies are diffcult due to the confounding variables of type and mechanism of injury, severity of injury, and length of time to present to emergency departments. While animal models exist, it is our goal to develop an in-vitro model of TBI using primary human brain cell cultures to examine cellular responses in real time. While the blood-brain barrier has been studied extensively post-TBI due to its role in ischemia and hypoxia, there is a dearth of research on the blood-CSF-barrier (BCSFB), the choroid plexus, and its response post-TBI. After culturing primary human choroid plexus endothelial cells (ScienCell Cat #1300) on a flexible membrane, we subjected them to mechanical strain using the FlexCell tension system and collected supernatant at 3-, 6-, 12-, 24-, 48-, 72-, and 96-hours post injury. The FlexCell tension bioreactor system uses positive and negative air pressure to apply mechanical elongation strain mimicking mild shearing injury. Using R&amp;D cytokine arrays (R&amp;D Systems, Cat# ARY005B), we measured 36 different cytokines, chemokines and acute phase proteins including C5a, GM-CSF, ICAM-1, IL-1, IL-6, TNFα, IFNγ, CCL5/RANTES, etc. We hypothesized that we would see a pro-inflammatory response (IL-1β, IL-6, TNFα) within 24 hours after one or two 12% mechanical strain injuries compared to the control of no strain, as seen in the literature. However, there were not significant increases in cytokine signaling until 24-48 hours post-injury, and in regulatory cytokines, such as CCL2, MIP1α/MIP1β, IL-2, IL-12, &amp; IL-17, that lasted as long as 96-hours. This effect was more pronounced in the two strain verses one strain model and control, as expected. Interestingly, anti-inflammatory cytokine IL-10 was significantly increased in the one strain model, but not two strain model, 48 hours post-insult. As the choroid plexus is a niche for T cell stimulation in the central nervous system (Strominger et al., 2018), these results indicate a complex immunological response that seems to favor T-Cell response over monocytes/macrophages. These experiments were completed with the lowest mechanical strain of the FlexCell system and future experiments are warranted to determine if a stronger mechanical strain is needed to produce the pro-inflammatory response seen in the literature in mild to severe models of TBI. This project was funded by a Senior Research Grant from the Indiana Academy of Science and from a Faculty Research Development Grant from Marian University College of Osteopathic Medicine. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CELLULAR PHENOTYPES OF HUMAN OCULAR MELANOMA IN CONTEXT OF TREATMENT SELECTION

The purpose of the study was to show the histopathological features of melanoma of the human choroid, as well as the structures surrounding the tumor, and their significance in predicting the outcome of the tumor for choosing a treatment method. The desire for organ-preserving non-invasive methods of tumor destruction leaves out of attention such risk factors as metastasis, localization and degree of damage to the structures surrounding uveal melanomas. To substantiate the unsafety of local treatment of melanomas, the morphological features of the cellular phenotypes of the tumor and the features of changes in the structures surrounding the tumor on sections of a human eye enucleated according to clinical indications were established. It has been established that the tumor contains not only the well-known spindle-shaped and epithelioid cells. The data obtained complement the information on the cellular phenotypes of ocular melanomas and dictate a revision of the generally accepted classification of these tumors, including fusiform, epithelioid and mixed types of malignant tissues in the structure of the human choroid, adopted in the seventh edition of the Cancer Staging Manual of the American Joint Committee on Cancer (Cancer Staging Manual) of the American Joint Committee on Cancer). Pathological changes were established in the tissues adjacent to the tumor, in the retina and vitreous body, which are responsible for the preservation of visual functions.

Olfactory Receptor OR2K2 Expression in Human Choroid Plexus as a Potential Marker in Early Sporadic Alzheimer's Disease.

Epithelial cells comprising the choroid plexus (CP) form a crucial barrier between the blood and the cerebrospinal fluid, thereby assuming a central position in brain homeostasis and signaling. Mounting evidence suggests that the impairment of CP function may be a significant contributor to Alzheimer's disease (AD) pathogenesis. CP function relies on the expression of specific receptors, and the potential involvement of olfactory receptors (ORs) and taste receptors (TASRs) in chemical surveillance within the CP is being investigated. Previous studies have implicated ORs and TASRs in neurodegenerative disorders like AD, although the direct evidence of their expression in the human CP remains to be established. In this study, we conducted a transcriptomic analysis encompassing eleven ORs and TASRs in the CP, comparing samples from healthy age-matched controls to those from patients with AD spanning Braak stages I to VI. Among these receptors, a striking finding emerged-OR2K2 exhibited robust expression, with a statistically significant upregulation noted at Braak stage I. Surprisingly, at the protein level, OR2K2 showed a significant decrease in both Braak stage I and VI. Additionally, we identified CP epithelial cells as the source of OR2K2 expression, where it colocalized with autophagy markers LC3 and p62. We postulate that OR2K2 could be subjected to degradation by autophagy in the early stages of AD, triggering a compensatory mechanism that leads to increased OR2K2 mRNA transcription. This study uncovers a potential role for OR2K2 in AD pathogenesis, offering a novel perspective on the intricate dynamics at play in this neurodegenerative disorder.

Methods to extract and analyze fluid from human pluripotent stem cell-derived choroid plexus organoids.

The choroid plexus (ChP) is a highly vascularized tissue lining the ventricular space of the brain. The ChP generates cerebrospinal fluid (CSF) and forms a protective barrier in the central nervous system (CNS). Recently, a three-dimensional human pluripotent stem cell (hPSC)-derived ChP organoid model has been developed. This model generates cystic structures that are filled with a fluid resembling CSF and are surrounded by an epithelial layer expressing ependymal choroid plexus-specific markers. Here we describe a method to generate these choroid plexus organoids using a commercial kit and methods to extract the CSF-like fluid for use in downstream analysis.

Transcriptome Analysis of Choroidal Endothelium Links Androgen Receptor Role to Central Serous Chorioretinopathy.

Central Serous Chorioretinopathy (CSCR) manifests as fluid accumulation between the neurosensory retina and the retinal pigment epithelium (RPE). Elevated levels of steroid hormones have been implicated in CSCR pathogenesis. This investigation aims to delineate the gene expression patterns of CSCR-associated risk and steroid receptors across human choroidal cell types and RPE cells to discern potential underlying mechanisms. This study utilized a comprehensive query of transcriptomic data derived from non-pathological human choroid and RPE cells. CSCR-associated genes such as PTPRB, CFH, and others are predominantly expressed in the choroidal endothelium as opposed to the RPE. The androgen receptor, encoded by the AR gene, demonstrates heightened expression in the macular endothelium compared to peripheral regions, unlike other steroid receptor genes. AR-expressing endothelial cells display an augmented responsiveness to Transforming growth factor beta (TGF-β), indicating a propensity towards endothelial to mesenchymal transition (endMT) transcriptional profiling. These results highlight the proclivity of CSCR to manifest primarily within the choroidal vasculature rather than the RPE, suggesting its categorization as a vascular eye disorder. This study accentuates the pivotal role of androgenic steroids, in addition to glucocorticoids. The observed linkage to TGF-β-mediated endMT provides a potential mechanistic insight into the disease's etiology.

Insights into human choroid melanocyte and stromal cells

Aims/Purpose: The human choroid is a complex highly vascular pigmented tissue between Bruch's membrane and the sclera. Its dynamic nature is important for eye homeostasis and for in pathology including macular degeneration and myopia. The stroma comprises heterogeneous cell populations including fibroblasts, melanocytes, immune cells, smooth muscle cells and neurons within a loose collagenous extracellular matrix. We used RNA transcriptome and multimarker immunolabelling of human choroidal melanocytes and tissue to further characterize adult choroidal melanocytes.Methods: Human choroidal melanocytes were cultured (n = 6 postmortem donor eyes, &lt;18 h delay, UNSW HREC), and used between passage (P) 2 to 5 for RNA transcriptome and multimarker immunofluorescence labelling and confocal microscopy. Sections and flatmounts from central choroid were also compared. Immunolabelling included melanocyte (Tyrosinase, TRYP1, DCT, MelanA) and stromal cell (S1004A, vimentin, α‐smooth muscle actin, CD45) markers.Results: Cultured melanocytes displayed mostly bipolar morphology with heterogeneous eumelanin density related to donor eye iris colour. Whole transcriptome findings showed enriched melanogenesis genes (high level TYR, PMEL, MITF, TYRP1; MLANA; low level DCT1, MC1R) but not genes reported for RPE (e.g. RPE65), pericytes (e.g. ACTA2) or fibroblasts (e.g. IGF2) confirming melanocyte culture quality. We also found expression for proposed Schwann cell precursors PLP1 and SOX10. Immunolabelling confirmed overall expression of melanogenesis proteins, and subpopulations expressing PLP1 and Sox10, in culture and in choroidal tissue.Conclusions: Melanocytes and stromal fibroblast are among the most numerous and complex heterogeneous populations within the choroid and our study provides further details on characteristics markers and topography. A possible role for Schwann cell precursors in renewing adult melanocyte populations is intriguing and continues to be investigated.

Choroid plexus protein turnover in human choroid plexus organoids recapitulates turnover in humans measured using stable isotope labeling kinetics (SILK)

AbstractBackgroundThe choroid plexus secretes protein‐rich cerebrospinal fluid (CSF). CSF turnover is a key mechanism for protein clearance. Neurodegenerative diseases such as AD are characterised by accumulation of excess proteins that form pathological inclusions. CSF flow dysregulation could therefore be involved in the pathophysiology of neurodegenerative diseases (ND) such as Alzheimer’s Disease. A method for measuring CSF turnover in vivo may be achieved by quantitating CSF derived protein kinetics, providing a biomarker tool with to interrogate the relationship between CSF flow disequilibrium and neurodegeneration.MethodUsing untargeted proteomics we identify the most abundant proteins in a) stem‐cell derived choroid plexus organoids and b) human CSF. We then develop targeted mass spectrometry methods to quantitate protein specific peptides in both matrices. Using a stable isotope (13C6 leucine) we label a) choroid plexus organoids and b) humans with suspected NPH undergoing a 72 hour lumbar drainage protocol. We collect serial samples of the CSF‐like organoid fluid and CSF. We then measure the ratio of labelled to unlabelled peptides to quantitate the rates of protein labelling (fractional synthesis rate) and label loss (fractional clearance rate), defining the protein turnover rate.ResultHighly‐expressed proteins in human CSF from nine individuals were also present in CSF‐like fluid from CP organoids. Of these common proteins, we were able to quantitate the turnover rates of transthyretin, apolipoprotein E, cystatin C and pigment epithelium‐derived factor, and the synthesis rates of serotransferrin and albumin. We will correlate these findings with clinical measures of CSF production.ConclusionChoroid plexus protein synthesis and clearance can be measured using SILK; choroid plexus organoid fluid protein turnover recapitulates that seen in human CSF.

Choroid plexus mast cells drive tumor-associated hydrocephalus

Tumor-associated hydrocephalus (TAH) is a common and lethal complication of brain metastases. Although other factors beyond mechanical obstructions have been suggested, the exact mechanisms are unknown. Using single-nucleus RNA sequencing and spatial transcriptomics, we find that a distinct population of mast cells locate in the choroid plexus and dramatically increase during TAH. Genetic fate tracing and intracranial mast-cell-specific tryptase knockout showed that choroid plexus mast cells (CPMCs) disrupt cilia of choroid plexus epithelia via the tryptase-PAR2-FoxJ1 pathway and consequently increase cerebrospinal fluid production. Mast cells are also found in the human choroid plexus. Levels of tryptase in cerebrospinal fluid are closely associated with clinical severity of TAH. BMS-262084, an inhibitor of tryptase, can cross the blood-brain barrier, inhibit TAH invivo, and alleviate mast-cell-induced damage of epithelial cilia in a human pluripotent stem-cell-derived choroid plexus organoid model. Collectively, we uncover the function of CPMCs and provide an attractive therapy for TAH.

SARS-CoV-2 infects epithelial cells of the blood-cerebrospinal fluid barrier rather than endothelial cells or pericytes of the blood-brain barrier

BackgroundAs a consequence of SARS-CoV-2 infection various neurocognitive and neuropsychiatric symptoms can appear, which may persist for several months post infection. However, cell type-specific routes of brain infection and underlying mechanisms resulting in neuroglial dysfunction are not well understood.MethodsHere, we investigated the susceptibility of cells constituting the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP) to SARS-CoV-2 infection using human induced pluripotent stem cell (hiPSC)-derived cellular models and a ChP papilloma-derived epithelial cell line as well as ChP tissue from COVID-19 patients, respectively.ResultsWe noted a differential infectibility of hiPSC-derived brain microvascular endothelial cells (BMECs) depending on the differentiation method. Extended endothelial culture method (EECM)-BMECs characterized by a complete set of endothelial markers, good barrier properties and a mature immune phenotype were refractory to SARS-CoV-2 infection and did not exhibit an activated phenotype after prolonged SARS-CoV-2 inoculation. In contrast, defined medium method (DMM)-BMECs, characterized by a mixed endothelial and epithelial phenotype and excellent barrier properties were productively infected by SARS-CoV-2 in an ACE2-dependent manner. hiPSC-derived brain pericyte-like cells (BPLCs) lacking ACE2 expression were not susceptible to SARS-CoV-2 infection. Furthermore, the human choroid plexus papilloma-derived epithelial cell line HIBCPP, modeling the BCSFB was productively infected by SARS-CoV-2 preferentially from the basolateral side, facing the blood compartment. Assessment of ChP tissue from COVID-19 patients by RNA in situ hybridization revealed SARS-CoV-2 transcripts in ChP epithelial and ChP stromal cells.ConclusionsOur study shows that the BCSFB of the ChP rather than the BBB is susceptible to direct SARS-CoV-2 infection. Thus, neuropsychiatric symptoms because of COVID-19 may rather be associated with dysfunction of the BCSFB than the BBB. Future studies should consider a role of the ChP in underlying neuropsychiatric symptoms following SARS-CoV-2 infection.

Engineering choroid plexus-on-a-chip with oscillatory flow for modeling brain metastasis.

The human brain choroid plexus (ChP) is a highly organized secretory tissue with a complex vascular system and epithelial layers in the ventricles of the brain. The ChP is the body's principal source of cerebrospinal fluid (CSF); it also functions as a barrier to separate the blood from CSF, because the movement of CSF through the body is pulsatile in nature. Thus far, it has been challenging to recreate the specialized features and dynamics of the ChP in a physiologically relevant microenvironment. In this study, we recapitulated the ChP structure by developing a microfluidic chip in accordance with established design rules. Furthermore, we used image processing and analysis to mimic CSF flow dynamics within a rlcking system; we also used a hydrogel containing laminin to mimic brain extracellular matrix (ECM). Human ChP cells were cultured in the ChP-on-a-chip with in vivo-like CSF dynamic flow and an engineered ECM. The key ChP characteristics of capillaries, the epithelial layer, and secreted components were recreated in the adjusted microenvironment of our human ChP-on-a-chip. The drug screening capabilities of the device were observed through physiologically relevant drug responses from breast cancer cells that had spread in the ChP. ChP immune responses were also recapitulated in this device, as demonstrated by the motility and cytotoxic effects of macrophages, which are the most prevalent immune cells in the ChP. Our human ChP-on-a-chip will facilitate the elucidation of ChP pathophysiology and support the development of therapeutics to treat cancers that have metastasized into the ChP.

In Memoriam: Prof. Valerii Viktorovych Vit

The legendary professor Valerii Viktorovych Vit passed away on April 22, 2023. He was born in Odesa in 1948. After graduating from the Faculty of General Medicine, Odesa Medical Institute, in 1972, Vit worked some time as a dissector at Odesa Regional Hospital. He began his science career at the Filatov Institute of Eye Diseases and Tissue Therapy in 1975 as a junior research associate at the Pathomorphology Laboratory, became head of the laboratory in 1978 and was promoted to the Deputy Director for Science at the institute in 1991. Valerii Viktorovych Vit defended his Cand Sc (Med) thesis in 1978, and doctoral (Dr Sc (Med)) dissertation, The Pathological Anatomy and Therapeutic Pathomorphosis of Human Pigmented Uveal Tumors, in 1986. The dissertation research was conducted under Professor V.V. Voino-Yasenetskyi, an outstanding scientist, and focused on the morphogenesis and malignancy of human choroidal and ciliary tumors. Valerii Viktorovych Vit was promoted to professorship in 1989. He authored or co-authored about 400 papers and five monographs on visual system structure and the pathological anatomy of eye disease. Five individuals completed their Dr Sc (Med) degrees, and 18, their Cand Sc (Med) degrees, under the supervision or mentorship of Prof. Vit. He was the first to raise questions on the value of ocular oncology studies on (a) the transformation of an eye tumor in the course of treatment and (b) the efficacy of the therapy used for treatment. He will be remembered as an exceptionally talented doctor, scientist and manager of high competence and great professional experience and a person with a strong commitment to work. By his pupils and colleagues, Professor Vit, a true professional in his field, will remain a cherished memory. We offer our condolences to his family and loved ones.