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Alzheimer's disease (AD) is a progressive neurological disorder and the leading cause of dementia. Despite significant efforts, treatment strategies targeting amyloid-β have been less successful than anticipated. Recently, the role of neuroinflammation and adaptive immune response in AD pathogenesis has gained attention. Here, we performed immune cell-enriched single-cell RNA sequencing of brain parenchymal cells from 12-month-old 5xFAD, an AD mouse model. We analyzed 11,587 single cells and found distinct differences in T cell and choroid plexus cell populations between 5xFAD mouse and littermate control. Subsequent sub-clustering of T cells in the 5xFAD mouse revealed distinct subtypes, with CD8+ resident memory T cells (T RM) being the most prevalent T cell type. In addition, we observed an increase in T cell exhaustion markers, including Pdcd1, Ctla4, and Havcr2 , with a particularly significant elevation of PD-1 and TIM-3 in CD8+ T RM in 5xFAD mouse. Furthermore, choroid plexus (ChP) epithelial cells showed altered gene expression patterns, with higher expression of MHC class I and Type I IFN-stimulated genes in 5xFAD mouse compared to the control mouse, suggesting an association with clonal expansion of AD-specific T cells in the brain. Through single-cell RNA sequencing (scRNA-seq) analysis, our study highlights the potential role of resident memory CD8+ T cell and their possible interactions with ChP epithelial cells. This study provides an exploration of the brain microenvironment landscape in AD, revealing critical insights into its underlying mechanisms. • Utilized single-cell RNA sequencing to delineate T cell changes in AD pathology. • CD8+ T RM cells emerged as the most prevalent subtype of T cells in the 5xFAD mouse brain. • Upregulation of exhaustion markers was observed in CD8+ T RM cells within the 5xFAD mouse brain. • Choroid plexus epithelial cells in AD show upregulated MHC class I expression. [ABSTRACT FROM AUTHOR]

The concept of a morphogen - a molecule that specifies two or more cell fates in a concentration-dependent manner - is paradigmatic in developmental biology. Much remains unknown, however, about the existence of morphogens in the developing vertebrate central nervous system (CNS), including the mouse dorsal telencephalic midline (DTM). Bone morphogenetic proteins (BMPs) are candidate DTM morphogens, and our previous work demonstrated BMP4 sufficiency to induce one DTM cell fate - that of choroid plexus epithelial cells (CPECs) - in a mouse embryonic stem cell (mESC) culture system. Here we used BMP4 in a modified mESC culture system to derive a second DTM fate, the cortical hem (CH). CH and CPEC markers were induced by BMP4 in a concentration-dependent manner consistent with in vivo development. BMP4 concentrations that led to CH fate also promoted markers for Cajal-Retzius neurons, which are known CH derivatives. Interestingly, single BMP4 administrations also sufficed for appropriate temporal regulation of CH, CPEC, and cortical genes, with initially broad and overlapping dose-response profiles that sharpened over time. BMP4 concentrations that yielded CH- or CPEC-enriched populations also had different steady-state levels of phospho-SMAD1/5/8, suggesting that differences in BMP signaling intensity underlie DTM fate choice. Surprisingly, inactivation of the cortical selector gene Lhx2 did not affect DTM expression levels, dose-response profiles, or timing in response to BMP4, although neural progenitor genes were downregulated. These data indicate that BMP4 can act as a classic morphogen to orchestrate both spatial and temporal aspects of DTM fate acquisition, and can do so in the absence of Lhx2.

Abstract Background Choroid plexus (CP) is an important tissue not only to produce cerebrospinal fluid (CSF) but also to regulate substances that are secreted into or absorbed from CSF through blood–cerebrospinal fluid barrier (BCSFB) formed by CP epithelial cells (CPECs). CPECs display signs of deterioration in aged and diseased people. However, whether CPECs in hypercholesterolemic animals develop such damage is not known. Methods We used cholesterol-fed wild-type or Watanabe hereditary hyperlipidemic (WHHL) rabbits of identical age to determine CPEC changes in terms of morphology and protein expression/localization. Results Compared with non-cholesterol-fed control rabbits, prolonged exposure to cholesterol reduced CPEC height and increased lipofuscin levels in CPECs, indicating cellular damage. Expression of aquaporin 1 on the apical membranes of CPECs was diminished in cholesterol-exposed rabbits, implying a reduced CSF-producing function in the CP. The rabbit macrophage-specific antibody (RAM11) immunoreaction became positive in CPECs adjacent to foam cells, indicating an alteration in this cell type. Conclusion Cholesterol insults from the circulation (which is reflected by foam-cell accumulation in the CP) induce CPEC dysfunction, and the latter seems to be enhanced by foam cells in hypercholesterolemic rabbits.

Background: Choroid plexus (CP) is an important tissue not only to produce cerebrospinal fluid (CSF) but also to regulate substances that are secreted into or absorbed from CSF through blood–cerebrospinal fluid barrier (BCSFB) formed by CP epithelial cells (CPECs). CPECs display signs of deterioration in aged and diseased people. However, whether CPECs in hypercholesterolemic animals develop such damage is not known. Methods: We used cholesterol-fed wild-type or Watanabe hereditary hyperlipidemic (WHHL) rabbits of identical age to determine CPEC changes in terms of morphology and protein expression/localization. Results: Compared with non-cholesterol-fed control rabbits, prolonged exposure to cholesterol reduced CPEC height and increased lipofuscin levels in CPECs, indicating cellular damage. Expression of aquaporin 1 on the apical membranes of CPECs was diminished in cholesterol-exposed rabbits, implying a reduced CSF-producing function in the CP. The rabbit macrophage-specific antibody (RAM11) immunoreaction became positive in CPECs adjacent to foam cells, indicating an alteration in this cell type. Conclusion: Cholesterol insults from the circulation (which is reflected by foam-cell accumulation in the CP) induce CPEC dysfunction, and the latter seems to be enhanced by foam cells in hypercholesterolemic rabbits. [ABSTRACT FROM AUTHOR]

The concept of a morphogen – a molecule that specifies two or more cell fates in a concentration-dependent manner – is paradigmatic in developmental biology. Much remains unknown, however, about the existence of morphogens in the developing vertebrate central nervous system (CNS), including the mouse dorsal telencephalic midline (DTM). Bone morphogenetic proteins (BMPs) are candidate DTM morphogens, and our previous work demonstrated BMP4 sufficiency to induce one DTM cell fate – that of choroid plexus epithelial cells (CPECs) – in a mouse embryonic stem cell (mESC) culture system. Here we used BMP4 in a modified mESC culture system to derive a second DTM fate, the cortical hem (CH). CH and CPEC markers were induced by BMP4 in a concentration-dependent manner consistent with in vivo development. BMP4 concentrations that led to CH fate also promoted markers for Cajal–Retzius neurons, which are known CH derivatives. Interestingly, single BMP4 administrations also sufficed for appropriate temporal regulation of CH, CPEC, and cortical genes, with initially broad and overlapping dose-response profiles that sharpened over time. BMP4 concentrations that yielded CH- or CPEC-enriched populations also had different steady-state levels of phospho-SMAD1/5/8, suggesting that differences in BMP signaling intensity underlie DTM fate choice. Surprisingly, inactivation of the cortical selector gene Lhx2 did not affect DTM expression levels, dose-response profiles, or timing in response to BMP4, although neural progenitor genes were downregulated. These data indicate that BMP4 can act as a classic morphogen to orchestrate both spatial and temporal aspects of DTM fate acquisition, and can do so in the absence of Lhx2.

Transplantation of bone marrow stromal cells (BMSCs) enhanced the outgrowth of regenerating axons and promoted locomotor improvements of rats with spinal cord injury (SCI). BMSCs did not survive long-term, disappearing from the spinal cord within 2-3 weeks after transplantation. Astrocyte-devoid areas, in which no astrocytes or oligodendrocytes were found, formed at the epicenter of the lesion. It was remarkable that numerous regenerating axons extended through such astrocyte-devoid areas. Regenerating axons were associated with Schwann cells embedded in extracellular matrices. Transplantation of choroid plexus epithelial cells (CPECs) also enhanced axonal regeneration and locomotor improvements in rats with SCI. Although CPECs disappeared from the spinal cord shortly after transplantation, an extensive outgrowth of regenerating axons occurred through astrocyte-devoid areas, as in the case of BMSC transplantation. These findings suggest that BMSCs and CPECs secret neurotrophic factors that promote tissue repair of the spinal cord, including axonal regeneration and reduced cavity formation. This means that transplantation of BMSCs and CPECs promotes "intrinsic" ability of the spinal cord to regenerate. The treatment to stimulate the intrinsic regeneration ability of the spinal cord is the safest method of clinical application for SCI. It should be emphasized that the generally anticipated long-term survival, proliferation and differentiation of transplanted cells are not necessarily desirable from the clinical point of view of safety.

Transplantation of somatic cells, including bone marrow stromal cells (BMSCs), bone marrow mononuclear cells (BMNCs), and choroid plexus epithelial cells (CPECs), enhances the outgrowth of regenerating axons and promotes locomotor improvements. They are not integrated into the host spinal cord, but disappear within 2-3 weeks after transplantation. Regenerating axons extend at the spinal cord lesion through the astrocyte-devoid area that is filled with connective tissue matrices. Regenerating axons have characteristics of peripheral nerves: they are associated with Schwann cells, and embedded in connective tissue matrices. It has been suggested that neurotrophic factors secreted from BMSCs and CPECs promote “intrinsic” ability of the spinal cord to regenerate. Transplanted Schwann cells survive long-term, and are integrated into the host spinal cord, serving as an effective scaffold for the outgrowth of regenerating axons in the spinal cord. The disadvantage that axons are blocked to extend through the glial scar at the border of the lesion is overcome. Schwann cells have been approved for clinical applications. Neural stem/progenitor cells (NSPCs) survive long-term, proliferate, and differentiate into glial cells and/or neurons after transplantation. No method is available at present to manipulate and control the behaviors of NPSCs to allow them to appropriately integrate into the host spinal cord. NPSP transplantation is not necessarily effective for locomotor improvement.

Purpose: The effect of the transplantation of choroid plexus epithelial cells (CPECs) on locomotor improvement and tissue repair including axonal extension in spinal cord lesions was examined in rats with spinal cord injury (SCI). Methods: CPECs were cultured from the choroid plexus of green fluorescent protein (GFP)-transgenic rats, and transplanted directly into the contusion-injured spinal cord lesions of rats of the same strain. Locomotor behaviors were evaluated based on BBB scores every week after transplantation until 4 weeks after transplantation. Histological and immunohistochemical examinations were performed at 2 days, and every week until 5 weeks after transplantation. Results: Locomotor behaviors evaluated by the BBB score were significantly improved in cell-transplanted rats. Numerous axons grew, with occasional interactions with CPECs, through the astrocyte-devoid areas. These axons exhibited structural characteristics of peripheral nerves. GAP-43-positive axons were found at the border of the lesion 2 days after transplantation. Cavity formation was more reduced in cell-transplanted than control spinal cords. CPECs were found within the spinal cord lesion, and sometimes in association with astrocytes at the border of the lesion until 2 weeks after transplantation. Conclusion: The transplantation of CPECs enhanced locomotor improvement and tissue recovery, including axonal regeneration, in rats with SCI. [ABSTRACT FROM AUTHOR]

Abstract: Choroid plexus (CP) epithelial cells (CPECs) produce cerebrospinal fluid (CSF) to provide the CNS with a specialized microenvironment. Our previous study showed that the conditioned medium of cultured CPECs enhanced the survival and neurite extension of hippocampal neurons. The present study examined the ability of cultured CPECs to protect against ischemic brain injury when transplanted into the CSF. Rats were subjected to a transient occlusion of the middle cerebral artery, followed by an injection of cultured CPECs into the fourth ventricle. The injection markedly reduced neurological deficits and infarction volume within 24h. Other beneficial effects were (1) a reduction in number of apoptotic and inflammatory cells, (2) an up-regulation of the mRNA expression of an anti-apoptotic effecter, cAMP-response element binding protein, and (3) a down-regulation of the production of pro-inflammatory factors such as interleukin-1 beta and inducible nitric oxide synthase. The injected CPECs were located within the ventricles and on the brain''s surface, not in the ischemic foci, suggesting that they exert their effects by releasing diffusible neuroprotective factors into the CSF. The transplantation of CPECs via CSF is a potential new strategy for protecting against ischemic brain injury. [Copyright &y& Elsevier]

The blood–cerebrospinal fluid barrier (BCSFB) plays a key role in the influx and efflux transport of drugs and endogenous substrates in the cerebrospinal fluid (CSF). To clarify the molecular mechanism of the BCSFB transport system, a new in vitro BCSFB model, i.e. an immortalized rat choroid plexus epithelial cell line (TR-CSFB), has been established from transgenic rats harboring a temperature-sensitive simian virus 40 large T-antigen gene. TR-CSFB cells grow well at 33 °C because of activation of the temperature-sensitive large T-antigen. These cells have a polygonal epithelial cell morphology and express typical choroid plexus epithelial cell markers, such as transthyretin (TTR) and Na+, K+-ATPase, as well as the transporters, system A and ABCC1/mrp1. The localization of Na+, K+-ATPase, and the transport direction of system A are polarized in TR-CSFB cells as is the case in vivo. TR-CSFB cells exhibit l-proline and l-glutamic acid uptake activities and may reflect the CSF-to-blood efflux transport functions involving these amino acids in vivo. Using TR-CSFB cells, we found for the first time that oatp3 is expressed at the BCSFB. TR-CSFB cells appear to be a useful in vitro model of the BCSFB for the study of drug transport, BCSFB transporters, and the regulation of BCSFB functions. [Copyright &y& Elsevier]

Crossing the blood–CSF barrier is an important pathway for certain nutrients to enter the CNS. Cultured choroid plexus epithelial cells are a potent model system to study active transport properties of this tissue in vitro. In the present study this in vitro model was used to analyse ascorbic acid transport across the blood–CSF barrier that is supposedly mediated by the Na+-dependent transporter SVCT2. The expression of SVCT2 in the cultured cells was proven by RT-PCR. Active transport across the cell monolayer resulted in ascorbic acid enrichment at the CSF mimicking side. Ascorbic acid transport and uptake were decreased to 13 and 27%, respectively, in the presence of 200 μM phloretin. Inhibition of both transepithelial substrate transport (to 7.5%) and cytoplasmatic uptake (to 20%) was observed in Na+-free medium indicating that a basolaterally located and Na+-dependent transporter mediates ascorbic acid uptake. Substituting Cl− by either iodide or d-gluconate increased ascorbic acid uptake by factors of 3.7 or 2.5, respectively. Similar observations were made when Na+-dependent myo-inositol transport was analysed. Additionally, in presence of 100 μM bumetanide, an inhibitor of Na+-Cl−-cotransport, indirectly increased ascorbic acid and myo-inositol transport rates were observed showing that ascorbic acid-Na+-cotransport might balance low intracellular Na+ concentration. [Copyright &y& Elsevier]

Purpose. To establish and characterize a choroid plexus epithelial cell line (TR-CSFB) from a new type of transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat). Methods. Choroid plexus epithelial cells were isolated from the Tg rat and cultured on a collagen-coated dish at 37°C during the first period of 3 days. Cells were subsequently cultured at 33°C to activate large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells using a penicillin cup. Results. Five immortalized cell lines of choroid plexus epithelial cells (TR-CSFB 1∼5) were obtained from two Tg rats. These cell lines had a polygonal cell morphology, expressed the typical choroid plexus epithelial cell marker, transthyretin, and possessed Na+, K+-ATPase on their apical side. TR-CSFBs cells expressed a large T-antigen and grew well at 33°C with a doubling-time of 35∼40 hr. [3H]-L-Proline uptake by TR-CSFB cells took place in an Na+-dependent, ouabain-sensitive, energy-dependent, and concentration-dependent manner. It was also inhibited by α-methylaminoisobutylic acid, suggesting that system A for amino acids operates in TR-CSFB cells. When [3H]-L-proline uptake was measured using the Transwell device, the L-proline uptake rate following application to the apical side was fivefold greater than that following application to the basal side. In addition, both Na+-dependent and Na+-independent L-glutamic acid (L-Glu) uptake processes were present in TR-CSFB cells. Conclusions. Immortalized choroid plexus epithelial cell lines were successfully established from Tg rats and have the properties of choroid plexus epithelial cells, and amino acid transport activity was observed in vivo. [ABSTRACT FROM AUTHOR]

Following an intraventricular hemorrhage (IVH), red blood cell lysis and hemoglobin (Hb) oxidation with the release of heme can cause sterile neuroinflammation. In this study, we measured Hb derivates and cellular adhesion molecules ICAM-1 and VCAM-1 with cell-free miRNAs in cerebrospinal fluid (CSF) samples obtained from Grade-III and Grade-IV preterm IVH infants (IVH-III and IVH-IV, respectively) at multiple time points between days 0–60 after the onset of IVH. Furthermore, human choroid plexus epithelial cells (HCPEpiCs) were incubated with IVH and non-IVH CSF (10 v/v %) for 24 h in vitro to investigate the IVH-induced inflammatory response that was investigated via: (i) HMOX1, IL8, VCAM1, and ICAM1 mRNAs as well as miR-155, miR-223, and miR-181b levels by RT-qPCR; (ii) nuclear translocation of the NF-κB p65 subunit by fluorescence microscopy; and (iii) reactive oxygen species (ROS) measurement. We found a time-dependent alteration of heme, IL-8, and adhesion molecules which revealed a prolonged elevation in IVH-IV vs. IVH-III with higher miR-155 and miR-181b expression at days 41–60. Exposure of HCPEpiCs to IVH CSF samples induced HMOX1, IL8, and ICAM1 mRNA levels along with increased ROS production via the NF-κB pathway activation but without cell death, as confirmed by the cell viability assay. Additionally, the enhanced intracellular miR-155 level was accompanied by lower miR-223 and miR-181b expression in HCPEpiCs after CSF treatment. Overall, choroid plexus epithelial cells exhibit an abnormal cell phenotype after interaction with pro-inflammatory CSF of IVH origin which may contribute to the development of later clinical complications in preterm IVH. [ABSTRACT FROM AUTHOR]

Transplantation of bone marrow stromal cells (BMSCs) enhanced the outgrowth of regenerating axons and promoted locomotor improvements of rats with spinal cord injury (SCI). BMSCs did not survive long-term, disappearing from the spinal cord within 2-3 weeks after transplantation. Astrocyte-devoid areas, in which no astrocytes or oligodendrocytes were found, formed at the epicenter of the lesion. It was remarkable that numerous regenerating axons extended through such astrocyte-devoid areas. Regenerating axons were associated with Schwann cells embedded in extracellular matrices. Transplantation of choroid plexus epithelial cells (CPECs) also enhanced axonal regeneration and locomotor improvements in rats with SCI. Although CPECs disappeared from the spinal cord shortly after transplantation, an extensive outgrowth of regenerating axons occurred through astrocyte-devoid areas, as in the case of BMSC transplantation. These findings suggest that BMSCs and CPECs secret neurotrophic factors that promote tissue repair of the spinal cord, including axonal regeneration and reduced cavity formation. This means that transplantation of BMSCs and CPECs promotes "intrinsic" ability of the spinal cord to regenerate. The treatment to stimulate the intrinsic regeneration ability of the spinal cord is the safest method of clinical application for SCI. It should be emphasized that the generally anticipated long-term survival, proliferation and differentiation of transplanted cells are not necessarily desirable from the clinical point of view of safety., Competing Interests: Conflicts of Interest: None declared.

Transplantation of somatic cells, including bone marrow stromal cells (BMSCs), bone marrow mononuclear cells (BMNCs), and choroid plexus epithelial cells (CPECs), enhances the outgrowth of regenerating axons and promotes locomotor improvements. They are not integrated into the host spinal cord, but disappear within 2-3 weeks after transplantation. Regenerating axons extend at the spinal cord lesion through the astrocyte-devoid area that is filled with connective tissue matrices. Regenerating axons have characteristics of peripheral nerves: they are associated with Schwann cells, and embedded in connective tissue matrices. It has been suggested that neurotrophic factors secreted from BMSCs and CPECs promote "intrinsic" ability of the spinal cord to regenerate. Transplanted Schwann cells survive long-term, and are integrated into the host spinal cord, serving as an effective scaffold for the outgrowth of regenerating axons in the spinal cord. The disadvantage that axons are blocked to extend through the glial scar at the border of the lesion is overcome. Schwann cells have been approved for clinical applications. Neural stem/progenitor cells (NSPCs) survive long-term, proliferate, and differentiate into glial cells and/or neurons after transplantation. No method is available at present to manipulate and control the behaviors of NPSCs to allow them to appropriately integrate into the host spinal cord. NPSP transplantation is not necessarily effective for locomotor improvement., Competing Interests: Conflicts of Interest: None declared.

Purpose: The effect of the transplantation of choroid plexus epithelial cells (CPECs) on locomotor improvement and tissue repair including axonal extension in spinal cord lesions was examined in rats with spinal cord injury (SCI)., Methods: CPECs were cultured from the choroid plexus of green fluorescent protein (GFP)-transgenic rats, and transplanted directly into the contusion-injured spinal cord lesions of rats of the same strain. Locomotor behaviors were evaluated based on BBB scores every week after transplantation until 4 weeks after transplantation. Histological and immunohistochemical examinations were performed at 2 days, and every week until 5 weeks after transplantation., Results: Locomotor behaviors evaluated by the BBB score were significantly improved in cell-transplanted rats. Numerous axons grew, with occasional interactions with CPECs, through the astrocyte-devoid areas. These axons exhibited structural characteristics of peripheral nerves. GAP-43-positive axons were found at the border of the lesion 2 days after transplantation. Cavity formation was more reduced in cell-transplanted than control spinal cords. CPECs were found within the spinal cord lesion, and sometimes in association with astrocytes at the border of the lesion until 2 weeks after transplantation., Conclusion: The transplantation of CPECs enhanced locomotor improvement and tissue recovery, including axonal regeneration, in rats with SCI.