Meningeal B Cell Clusters Correlate with Submeningeal Pathology in a Natural Model of Multiple Sclerosis.

Multiple sclerosis (MS) is an idiopathic demyelinating disease in which meningeal inflammation correlates with accelerated disease progression. The study of meningeal inflammation in MS has been limited because of constrained access to MS brain/spinal cord specimens and the lack of experimental models recapitulating progressive MS. Unlike induced models, a spontaneously occurring model would offer a unique opportunity to understand MS immunopathogenesis and provide a compelling framework for translational research. We propose granulomatous meningoencephalomyelitis (GME) as a natural model to study neuropathological aspects of MS. GME is an idiopathic, progressive neuroinflammatory disease of young dogs with a female bias. In the GME cases examined in this study, the meninges displayed focal and disseminated leptomeningeal enhancement on magnetic resonance imaging, which correlated with heavy leptomeningeal lymphocytic infiltration. These leptomeningeal infiltrates resembled tertiary lymphoid organs containing large B cell clusters that included few proliferating Ki67+ cells, plasma cells, follicular dendritic/reticular cells, and germinal center B cell-like cells. These B cell collections were confined in a specialized network of collagen fibers associated with the expression of the lympho-organogenic chemokines CXCL13 and CCL21. Although neuroparenchymal perivascular infiltrates contained B cells, they lacked the immune signature of aggregates in the meningeal compartment. Finally, meningeal B cell accumulation correlated significantly with cortical demyelination reflecting neuropathological similarities to MS. Hence, during chronic neuroinflammation, the meningeal microenvironment sustains B cell accumulation that is accompanied by underlying neuroparenchymal injury, indicating GME as a novel, naturally occurring model to study compartmentalized neuroinflammation and the associated pathology thought to contribute to progressive MS.

Ovariectomy Influences Cognition and Markers of Alzheimer's Disease.

Alzheimer's disease (AD) is one of the most devastating and costly diseases, and prevalence of AD increases with age. Furthermore, females are twice as likely to suffer from AD compared to males. The cessation of reproductive steroid hormone production during menopause is hypothesized to cause this difference. Two rodent AD models, APP21 and APP+PS1, and wild type (WT) rats underwent an ovariectomy or sham surgery. Changes in learning and memory, brain histology, amyloid-ß (Aß) deposition, levels of mRNAs involved in Aß production and clearance, and synaptic and cognitive function were determined. Barnes maze results showed that regardless of ovariectomy status, APP+PS1 rats learned slower and had poor memory retention. Ovariectomy caused learning impairment only in the APP21 rats. High levels of Aß42 and very low levels of Aß40 were observed in the brain cortices of APP+PS1 rats indicating limited endogenous PS1. The APP+PS1 rats had 43-fold greater formic acid soluble Aß42 than Aß40 at 17 months. Furthermore, levels of formic acid soluble Aß42 increased 57-fold in ovariectomized APP+PS1 rats between 12 and 17 months of age. The mRNA encoding Grin1 significantly decreased due to ovariectomy whereas levels of Bace1, Chat, and Prkcb all decreased with age. The expression levels of mRNAs involved in Aß degradation and AßPP cleavage (Neprilysin, Ide, Adam9, and Psenen) were found to be highly correlated with each other as well as hippocampal Aß deposition. Taken together, these results indicate that both ovariectomy and genotype influence AD markers in a complex manner.

Ontogeny of inter-alpha inhibitor protein (IAIP) expression in human brain.

Inter-alpha inhibitor proteins (IAIPs) are naturally occurring immunomodulatory molecules found in most tissues. We have reported ontogenic changes in the expression of IAIPs in brain during development in sheep and abundant expression of IAIPs in fetal and neonatal rodent brain in a variety of cellular types and brain regions. Although a few studies identified bikunin, light chain of IAIPs, in adult human brain, the presence of the complete endogenous IAIP protein complex has not been reported in human brain. In this study, we examined the immunohistochemical expression of endogenous IAIPs in human cerebral cortex from early in development through the neonatal period and in adults using well-preserved postmortem brains. We examined total, nuclear, and cytoplasmic staining of endogenous IAIPs and their expression in neurofilament light polypeptide-positive neurons and glial fibrillary acidic protein (GFAP)-positive astrocytes. IAIPs were ubiquitously detected for the first time in cerebral cortical cells at 24-26, 27-28, 29-36, and 37-40 weeks of gestation and in adults. Quantitative analyses revealed that IAIPs were predominately localized in the nucleus in all age groups, but cytoplasmic IAIP expression was more abundant in adult than in the younger ages. Immunoreactivity of IAIPs was expressed in neurons and astrocytes in all age groups. In addition, IAIP co-localization with GFAP-positive astrocytes was more abundant in adults than in the developing brain. We conclude that IAIPs exhibit ubiquitous expression, and co-localize with neurons and astrocytes in the developing and adult human brain suggesting a potential role for IAIPs in development and endogenous neuroprotection.

Enteric Pathologic Manifestations of Alpha-Synucleinopathies.

BACKGROUND: Gastrointestinal (GI) symptoms are common in Parkinson disease (PD), often preceding neurological manifestations; however, early diagnostic utility of GI biopsies remains controversial. Studies suggest aberrant deposition of alpha-synuclein (α-syn) follows step-wise progression in central nervous system though histologic interpretation of normal and aberrant staining patterns have shown variable results. This study examines whether GI α-syn mRNA expression combined with standard α-syn immunohistochemical staining enhance the role of GI biopsy in PD. MATERIALS AND METHODS: Four groups were examined, including pediatric (21) and adult control patients (18), PD clinic patients (17), and pathologically confirmed PD cases from hospital archives (16). Enteric nervous system α-syn staining was evaluated by immunohistochemistry in 33 PD and 39 controls. α-Syn mRNA levels were compared between patient groups using quantitative polymerase chain reaction and stomach and colon levels in PD. RESULTS: PD patients had Lewy bodies (LB) and diffuse neuronal α-syn staining. GI tissues from elderly controls, children, and young adults exhibited diffuse positivity. LB were limited to PD. Myenteric plexus immunoreactivity varied in different regions. Widespread staining was noted within stomach and colon. Immunoreactivity was present within esophagus, appendix, and small bowel. α-Syn mRNA expression was highest in PD; however, levels varied between proximal and distal GI tract. CONCLUSIONS: α-Syn is normally present within young and elderly enteric nervous system; furthermore, while α-syn mRNA is always detectable, levels are highest and most variable in PD. This suggests that enteric α-syn may be altered in neurodegenerative disease. The presence of LB in the GI tract, not solely α-syn expression, may prove useful, distinguishing neurodegenerative disease patients from normal controls.

Gastrointestinal iron excretion and reversal of iron excess in a mouse model of inherited iron excess.

The current paradigm in the field of mammalian iron biology states that body iron levels are determined by dietary iron absorption, not by iron excretion. Iron absorption is a highly regulated process influenced by iron levels and other factors. Iron excretion is believed to occur at a basal rate irrespective of iron levels and is associated with processes such as turnover of intestinal epithelium, blood loss, and exfoliation of dead skin. Here we explore iron excretion in a mouse model of iron excess due to inherited transferrin deficiency. Iron excess in this model is attributed to impaired regulation of iron absorption leading to excessive dietary iron uptake. Pharmacological correction of transferrin deficiency not only normalized iron absorption rates and halted progression of iron excess but also reversed body iron excess. Transferrin treatment did not alter the half-life of 59Fe in mutant mice. 59Fe-based studies indicated that most iron was excreted via the gastrointestinal tract and suggested that iron-loaded mutant mice had increased rates of iron excretion. Direct measurement of urinary iron levels agreed with 59Fe-based predictions that urinary iron levels were increased in untreated mutant mice. Fecal ferritin levels were also increased in mutant mice relative to wild-type mice. Overall, these data suggest that mice have a significant capacity for iron excretion. We propose that further investigation into iron excretion is warranted in this and other models of perturbed iron homeostasis, as pharmacological targeting of iron excretion may represent a novel means of treatment for diseases of iron excess.

Choroid plexus genes for CSF production and brain homeostasis are altered in Alzheimer's disease.

BACKGROUND: The roles of the choroid plexus (CP) and cerebrospinal fluid (CSF) production have drawn increasing attention in Alzheimer's disease (AD) research. Specifically, studies document markedly decreased CSF production and turnover in moderate-to-severe AD. Moreover, reduced CP function and CSF turnover lead to impaired clearance of toxic metabolites, likely promote neuroinflammation, and may facilitate neuronal death during AD progression. We analyzed CP gene expression in AD compared with control subjects, specifically considering those genes involved with CSF production and CP structural integrity. METHODS: The Brown-Merck Gene Expression Omnibus (GEO) database (CP transcripts) was mined to examine changes in gene expression in AD compared to controls with a focus on assorted genes thought to play a role in CSF production. Specifically, genes coding for ion transporters in CP epithelium (CPE) and associated enzymes like Na-K-ATPase and carbonic anhydrase, aquaporins, mitochondrial transporters/enzymes, blood-cerebrospinal fluid barrier (BCSFB) stability proteins, and pro-inflammatory mediators were selected for investigation. Data were analyzed using t test p-value and fold-change analysis conducted by the GEO2R feature of the GEO database. RESULTS: Significant expression changes for several genes were observed in AD CP. These included disruptions to ion transporters (e.g., the solute carrier gene SLC4A5, p = 0.004) and associated enzyme expressions (e.g., carbonic anhydrase CA4, p = 0.0001), along with decreased expression of genes involved in BCSFB integrity (e.g., claudin CLDN5, p = 0.039) and mitochondrial ATP synthesis (e.g., adenosine triphosphate ATP5L, p = 0.0004). Together all changes point to disrupted solute transport at the blood-CSF interface in AD. Increased expression of pro-inflammatory (e.g., interleukin IL1RL1, p = 0.00001) and potential neurodegenerative genes (e.g., amyloid precursor APBA3, p = 0.002) also implicate disturbed CP function. CONCLUSIONS: Because the altered expression of numerous transcripts in AD-CP help explain decreased CSF production in AD, these findings represent a first step towards identifying novel therapeutic targets in AD.

Blood-Cerebrospinal Fluid Barrier Gradients in Mild Cognitive Impairment and Alzheimer's Disease: Relationship to Inflammatory Cytokines and Chemokines.

Background: The pathophysiology underlying altered blood-cerebrospinal fluid barrier (BCSFB) function in Alzheimer's disease (AD) is unknown but may relate to endothelial cell activation and cytokine mediated inflammation. Methods: Cerebrospinal fluid (CSF) and peripheral blood were concurrently collected from cognitively healthy controls (N = 21) and patients with mild cognitive impairment (MCI) (N = 8) or AD (N = 11). The paired serum and CSF samples were assayed for a panel of cytokines, chemokines, and related trophic factors using multiplex ELISAs. Dominance analysis models were conducted to determine the relative importance of the inflammatory factors in relationship to BCSFB permeability, as measured by CSF/serum ratios for urea, creatinine, and albumin. Results: BCSFB disruption to urea, a small molecule distributed by passive diffusion, had a full model coefficient of determination (r2) = 0.35, and large standardized dominance weights (>0.1) for monocyte chemoattractant protein-1, interleukin (IL)-15, IL-1rα, and IL-2 in serum. BCSFB disruption to creatinine, a larger molecule governed by active transport, had a full model r2 = 0.78, and large standardized dominance weights for monocyte inhibitor protein-1b in CSF and tumor necrosis factor-α in serum. BCSFB disruption to albumin, a much larger molecule, had a full model r2 = 0.62, and large standardized dominance weights for IL-17a, interferon-gamma, IL-2, and VEGF in CSF, as well IL-4 in serum. Conclusions: Inflammatory proteins have been widely documented in the AD brain. The results of the current study suggest that changes in BCSFB function resulting in altered permeability and transport are related to expression of specific inflammatory proteins, and that the shifting distribution of these proteins from serum to CSF in AD and MCI is correlated with more severe perturbations in BCSFB function.

Counteracting the effects of TNF receptor-1 has therapeutic potential in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia, and neuroinflammation is an important hallmark of the pathogenesis. Tumor necrosis factor (TNF) might be detrimental in AD, though the results coming from clinical trials on anti-TNF inhibitors are inconclusive. TNFR1, one of the TNF signaling receptors, contributes to the pathogenesis of AD by mediating neuronal cell death. The blood-cerebrospinal fluid (CSF) barrier consists of a monolayer of choroid plexus epithelial (CPE) cells, and AD is associated with changes in CPE cell morphology. Here, we report that TNF is the main inflammatory upstream mediator in choroid plexus tissue in AD patients. This was confirmed in two murine AD models: transgenic APP/PS1 mice and intracerebroventricular (icv) AßO injection. TNFR1 contributes to the morphological damage of CPE cells in AD, and TNFR1 abrogation reduces brain inflammation and prevents blood-CSF barrier impairment. In APP/PS1 transgenic mice, TNFR1 deficiency ameliorated amyloidosis. Ultimately, genetic and pharmacological blockage of TNFR1 rescued from the induced cognitive impairments. Our data indicate that TNFR1 is a promising therapeutic target for AD treatment.

Succinate Dehydrogenase B (SDHB) Immunohistochemistry for the Evaluation of Muscle Biopsies.

Succinate dehydrogenase (SDH) is a key mitochondrial enzyme complex composed of 4 subunits. SDH histochemistry is routinely utilized in the assessment of muscle biopsies to reveal underlying pathology such as subsarcolemmal mitochondrial aggregates. In this study, we evaluated the utility of succinate dehydrogenase B (SDHB) immunohistochemistry (IHC) in 27 muscle biopsies, including 13 mitochondrial myopathies (MMs), 9 inflammatory myopathies, and 5 controls. SDHB IHC was performed on formalin-fixed, paraffin-embedded tissue sections with a mouse monoclonal antibody (Abcam 21A11AE7) in parallel with histochemical SDH stains on a fresh-frozen tissue. In all muscle biopsies, SDHB IHC exhibited granular immunoreactivity and highlighted the dark type 1 and lighter type 2 staining pattern observed by histochemistry. In all cases of MM, SDHB IHC showed subsarcolemmal granular aggregates involving the entire periphery of the fibers that were more distinct than those seen by SDH histochemistry. In 3 extraocular muscle biopsies, SDHB immunoreactive speckles of various sizes were distributed throughout the entire sarcoplasm that were more prominent than those seen on SDH histochemistry. Subsarcolemmal and cytoplasmic granular aggregates seen on SDHB IHC correlated with mitochondrial pathology on electron microscopy. In cases of inflammatory myopathy, there was diffuse sarcoplasmic SDHB immunoreactivity in degenerating fibers, but no evidence of subsarcolemmal aggregates. This study demonstrates that SDHB IHC is highly sensitive and specific in the identification of MM. The automation, reproducibility, and cost efficiency of SDHB IHC offer advantages over the labor-intensive histochemical method requiring frozen sections. As this technique is performed on formalin-fixed, paraffin-embedded tissues, it can be easily applied for retrospective studies.

The Orphan C2orf40 Gene is a Neuroimmune Factor in Alzheimer's Disease.

Expression of the orphan C2orf40 gene is associated with the aggregation of the neurofibrillary tangle-protein tau in transgenic mice, tumor suppression, the induction of senescence in CNS, and the activation of microglia and peripheral mononuclear leukocytes. This gene also encodes several secreted pro- and anti-inflammatory neuropeptide-like cytokines, suggesting they might be implicated in the inflammatory component(s) of Alzheimer's disease (AD). Accordingly, we evaluated human AD and control brains for expression changes by RT-qPCR, Western blot, and histological changes by immunolabeling. RT-qPCR demonstrated increased cortical gene expression in AD. The molecular form of Ecrg4 detected in cortex was 8-10 kDa, which was shown previously to interact with the innate immunity receptor complex. Immunocytochemical studies showed intensely stained microglia and intravascular blood-borne monocytes within cerebral cortical white matter of AD patients. Staining was diminished within cortical neurons, except for prominent staining in neurofibrillary tangles. Choroid plexuses showed a decreasing trend. These findings support our hypothesis that c2orf40 participates in the neuroimmune response in AD.

Atrophy of the tongue following complete versus partial hypoglossal nerve transection in a canine model.

OBJECTIVES/HYPOTHESIS: The hypoglossal nerve (XII) has been used as a donor nerve in facial and laryngeal reinnervation. The purpose of this study was to investigate the neuromuscular changes that occur within the tongue following partial or complete transection of XII using a canine model. STUDY DESIGN: Histopathological comparison of tongue denervation following two types of XII resection in a canine model. METHODS: Ten adult canines underwent complete unilateral resection of XII or resection of only the medial terminal branch of the hypoglossal nerve (mXII). After 6 months of recovery, tongue specimens were analyzed histopathologically using whole cross-sections. Routine histologic sections were assessed by two neuropathologists blinded to the type of denervation. The cross-sectional area was calculated of both sides of the tongue, and the amount of myosin was quantified morphometrically using immunohistochemistry for myosin (antimyosin heavy chain, fast isotype). Statistical comparison between partial and complete denervation was performed using the Student t test. RESULTS: Six months following XII transection, quantitative measures of the cross-sectional area of the tongue and content of myosin demonstrated severe muscle atrophy on the operated side of the tongue for both groups, compared to the nonoperated side. For partial transection involving only mXII, the degree of atrophy was less severe (P < .05). CONCLUSIONS: This study provides new histological information demonstrating that partial resection of the hypoglossal nerve, sacrificing only the proximal medial branch of the hypoglossal nerve (mXII), results in less severe atrophy of the tongue than complete transection of the entire hypoglossal nerve. LEVEL OF EVIDENCE: NA Laryngoscope, 126:2689-2693, 2016.

Human polyomavirus receptor distribution in brain parenchyma contrasts with receptor distribution in kidney and choroid plexus.

The human polyomavirus, JCPyV, is the causative agent of progressive multifocal leukoencephalopathy, a rare demyelinating disease that occurs in the setting of prolonged immunosuppression. After initial asymptomatic infection, the virus establishes lifelong persistence in the kidney and possibly other extraneural sites. In rare instances, the virus traffics to the central nervous system, where oligodendrocytes, astrocytes, and glial precursors are susceptible to lytic infection, resulting in progressive multifocal leukoencephalopathy. The mechanisms by which the virus traffics to the central nervous system from peripheral sites remain unknown. Lactoseries tetrasaccharide c (LSTc), a pentasaccharide containing a terminal α2,6-linked sialic acid, is the major attachment receptor for polyomavirus. In addition to LSTc, type 2 serotonin receptors are required for facilitating virus entry into susceptible cells. We studied the distribution of virus receptors in kidney and brain using lectins, antibodies, and labeled virus. The distribution of LSTc, serotonin receptors, and virus binding sites overlapped in kidney and in the choroid plexus. In brain parenchyma, serotonin receptors were expressed on oligodendrocytes and astrocytes, but these cells were negative for LSTc and did not bind virus. LSTc was instead found on microglia and vascular endothelium, to which virus bound abundantly. Receptor distribution was not changed in the brains of patients with progressive multifocal leukoencephalopathy. Virus infection of oligodendrocytes and astrocytes during disease progression is LSTc independent.

Ischemia/Reperfusion-induced neovascularization in the cerebral cortex of the ovine fetus.

Information on the effects of injury on neovascularization in the immature brain is limited. We investigated the effects of ischemia on cerebral cortex neovascularization after the exposure of fetuses to 30 minutes of cerebral ischemia followed by 48 hours of reperfusion (I/R-48), 30 minutes of cerebral ischemia followed by 72 hours of reperfusion (I/R-72), or sham control treatment (Non-I/R). Immunohistochemical and morphometric analyses of cerebral cortex sections included immunostaining for glial fibrillary acidic protein and collagen type IV (a molecular component of the vascular basal lamina) to determine the glial vascular network in fetal brains and Ki67 as a proliferation marker. Cerebral cortices from I/R-48 and I/R-72 fetuses exhibited general responses to ischemia, including reactive astrocyte morphology, which was not observed in Non-I/R fetuses. Cell bodies of reactive proliferating astrocytes, along with large end-feet, surrounded the walls of cerebral cortex microvessels in addition to the thick collagen type IV-enriched basal lamina. Morphometric analysis of the Non-I/R group with the I/R-48 and I/R-72 groups revealed increased collagen type IV density in I/R-72 cerebral cortex microvessels (p < 0.01), which also frequently displayed a sprouting appearance characterized by growing tip cells and activated pericytes. Increases in cerebral cortex basic fibroblast growth factor were associated with neovascularization. We conclude that increased neovascularization in fetal cerebral cortices occurs within 72 hours of ischemia.

Paclitaxel poliglumex, temozolomide, and radiation for newly diagnosed high-grade glioma: a Brown University Oncology Group Study.

OBJECTIVES: Paclitaxel poliglumex (PPX), a drug conjugate that links paclitaxel to poly-L-glutamic acid, is a potent radiation sensitizer. Prior studies in esophageal cancer have demonstrated that PPX (50 mg/m/wk) can be administered with concurrent radiation with acceptable toxicity. The primary objective of this study was to determine the safety of the combination of PPX with temozolomide and concurrent radiation for high-grade gliomas. METHODS: Eligible patients were required to have WHO grade 3 or 4 gliomas. Patients received weekly PPX (50 mg/m/wk) combined with standard daily temozolomide (75 mg/m) for 6 weeks with concomitant radiation (2.0 Gy, 5 d/wk for a total dose of 60 Gy). RESULTS: Twenty-five patients were enrolled, 17 with glioblastoma and 8 with grade 3 gliomas. Seven of 25 patients had grade 4 myelosuppression. Hematologic toxicity lasted up to 5 months suggesting a drug interaction between PPX and temozolomide. For patients with glioblastoma, the median progression-free survival was 11.5 months and the median overall survival was 18 months. CONCLUSIONS: PPX could not be safely combined with temozolomide due to grade 4 hematologic toxicity. However, the favorable progression-free and overall survival suggest that PPX may enhance radiation for glioblastoma. A randomized study of single agent PPX/radiation versus temozolomide/radiation for glioblastoma without MGMT methylation is underway.

Esophageal cancer related gene-4 is a choroid plexus-derived injury response gene: evidence for a biphasic response in early and late brain injury.

By virtue of its ability to regulate the composition of cerebrospinal fluid (CSF), the choroid plexus (CP) is ideally suited to instigate a rapid response to traumatic brain injury (TBI) by producing growth regulatory proteins. For example, Esophageal Cancer Related Gene-4 (Ecrg4) is a tumor suppressor gene that encodes a hormone-like peptide called augurin that is present in large concentrations in CP epithelia (CPe). Because augurin is thought to regulate senescence, neuroprogenitor cell growth and differentiation in the CNS, we evaluated the kinetics of Ecrg4 expression and augurin immunoreactivity in CPe after CNS injury. Adult rats were injured with a penetrating cortical lesion and alterations in augurin immunoreactivity were examined by immunohistochemistry. Ecrg4 gene expression was characterized by in situ hybridization. Cell surface augurin was identified histologically by confocal microscopy and biochemically by sub-cellular fractionation. Both Ecrg4 gene expression and augurin protein levels were decreased 24-72 hrs post-injury but restored to uninjured levels by day 7 post-injury. Protein staining in the supraoptic nucleus of the hypothalamus, used as a control brain region, did not show a decrease of auguin immunoreactivity. Ecrg4 gene expression localized to CPe cells, and augurin protein to the CPe ventricular face. Extracellular cell surface tethering of 14 kDa augurin was confirmed by cell surface fractionation of primary human CPe cells in vitro while a 6-8 kDa fragment of augurin was detected in conditioned media, indicating release from the cell surface by proteolytic processing. In rat CSF however, 14 kDa augurin was detected. We hypothesize the initial release and proteolytic processing of augurin participates in the activation phase of injury while sustained Ecrg4 down-regulation is dysinhibitory during the proliferative phase. Accordingly, augurin would play a constitutive inhibitory function in normal CNS while down regulation of Ecrg4 gene expression in injury, like in cancer, dysinhibits proliferation.

Alginate encapsulated human mesenchymal stem cells suppress syngeneic glioma growth in the immunocompetent rat.

Human mesenchymal stem cells (MSC) are promising candidates for cell therapy of neurological diseases. However, co-transplantation of MSC with tumour cell lines has been reported to promote tumour growth. In this study, we co-transplant glioma cells together with alginate-encapsulated MSC. Immunocompetent BD-IX rats were inoculated with syngeneic BT4Ca glioma cells. Encapsulated unmodified MSC, endostatin producing (endoMSC) or cell-free alginate capsules were stereotactically implanted into the tumour bed. After 12 days, tumour volumes were significantly diminished in the MSC-treated group. The decrease in tumour volume found with endoMSC was statistically not significant, despite significantly reduced tumour vascularization. We conclude that, under syngeneic conditions in the immunocompetent animal, (1) the intracranial, orthotopic co-transplantation of MSC with glioma cells leads to a suppression in tumour growth and (2) the tumour can escape the antiangiogenic treatment with endostatin. Our finding may facilitate the clinical translation of encapsulated cell therapy.

Ecrg4 expression and its product augurin in the choroid plexus: impact on fetal brain development, cerebrospinal fluid homeostasis and neuroprogenitor cell response to CNS injury.

BACKGROUND: The content and composition of cerebrospinal fluid (CSF) is determined in large part by the choroid plexus (CP) and specifically, a specialized epithelial cell (CPe) layer that responds to, synthesizes, and transports peptide hormones into and out of CSF. Together with ventricular ependymal cells, these CPe relay homeostatic signals throughout the central nervous system (CNS) and regulate CSF hydrodynamics. One new candidate signal is augurin, a newly recognized 14 kDa protein that is encoded by esophageal cancer related gene-4 (Ecrg4), a putative tumor suppressor gene whose presence and function in normal tissues remains unexplored and enigmatic. The aim of this study was to explore whether Ecrg4 and its product augurin, can be implicated in CNS development and the response to CNS injury. METHODS: Ecrg4 gene expression in CNS and peripheral tissues was studied by in situ hybridization and quantitative RT-PCR. Augurin, the protein encoded by Ecrg4, was detected by immunoblotting, immunohistochemistry and ELISA. The biological consequence of augurin over-expression was studied in a cortical stab model of rat CNS injury by intra-cerebro-ventricular injection of an adenovirus vector containing the Ecrg4 cDNA. The biological consequences of reduced augurin expression were evaluated by characterizing the CNS phenotype caused by Ecrg4 gene knockdown in developing zebrafish embryos. RESULTS: Gene expression and immunohistochemical analyses revealed that, the CP is a major source of Ecrg4 in the CNS and that Ecrg4 mRNA is predominantly localized to choroid plexus epithelial (CPe), ventricular and central canal cells of the spinal cord. After a stab injury into the brain however, both augurin staining and Ecrg4 gene expression decreased precipitously. If the loss of augurin was circumvented by over-expressing Ecrg4 in vivo, BrdU incorporation by cells in the subependymal zone decreased. Inversely, gene knockdown of Ecrg4 in developing zebrafish embryos caused increased proliferation of GFAP-positive cells and induced a dose-dependent hydrocephalus-like phenotype that could be rescued by co-injection of antisense morpholinos with Ecrg4 mRNA. CONCLUSION: An unusually elevated expression of the Ecrg4 gene in the CP implies that its product, augurin, plays a role in CP-CSF-CNS function. The results are all consistent with a model whereby an injury-induced decrease in augurin dysinhibits target cells at the ependymal-subependymal interface. We speculate that the ability of CP and ependymal epithelium to alter the progenitor cell response to CNS injury may be mediated, in part by Ecrg4. If so, the canonic control of its promoter by DNA methylation may implicate epigenetic mechanisms in neuroprogenitor fate and function in the CNS.

Targeting choroid plexus epithelia and ventricular ependyma for drug delivery to the central nervous system.

BACKGROUND: Because the choroid plexus (CP) is uniquely suited to control the composition of cerebrospinal fluid (CSF), there may be therapeutic benefits to increasing the levels of biologically active proteins in CSF to modulate central nervous system (CNS) functions. To this end, we sought to identify peptides capable of ligand-mediated targeting to CP epithelial cells reasoning that they could be exploited to deliver drugs, biotherapeutics and genes to the CNS. METHODS: A peptide library displayed on M13 bacteriophage was screened for ligands capable of internalizing into CP epithelial cells by incubating phage with CP explants for 2 hours at 37C and recovering particles with targeting capacity. RESULTS: Three peptides, identified after four rounds of screening, were analyzed for specific and dose dependent binding and internalization. Binding was deemed specific because internalization was prevented by co-incubation with cognate synthetic peptides. Furthermore, after i.c.v. injection into rat brains, each peptide was found to target phage to epithelial cells in CP and to ependyma lining the ventricles. CONCLUSION: These data demonstrate that ligand-mediated targeting can be used as a strategy for drug delivery to the central nervous system and opens the possibility of using the choroid plexus as a portal of entry into the brain.