archives@tulane.edu / The prevalence of HIV-associated neurocognitive disorders (HAND) has increased in the era of combination anti-retroviral therapy (cART). Despite this and documented neurocognitive impairment, there is a lack of pathology of HIV-encephalitis (HIVE), specifically multi-nucleated giant cells (MNGCs), in children and SIV-encephalitis (SIVE) in rhesus macaques infected pre-, peri-, and post-parturition. In this dissertation, we show that the lack of MNGCs seen is most likely due to innate differences in the blood-brain and blood-CSF barriers, and a robust pro- and anti-inflammatory response in neonatal rhesus macaques. Using a rhesus macaque model of HIV, we examined the plasma viral load, brain tissue viral load, and monocyte turnover, using PCR and flow cytometry, respectively. We also performed immunohistochemistry for monocyte, macrophage, tight junction, and aging markers of the choroid plexus. We sought to create a choroid plexus epithelial cell model to monitor the effects of inflammatory markers and virus on the tight junctions of the blood-CSF barrier in real-time. We demonstrated that neonates do not develop encephalitis, despite comparable viral load and monocyte turnover, previously established correlates of SIV-encephalitis (SIVE). However, we noted that uninfected adult rhesus macaques have an increase in virus susceptible cells in the brain, SIV-infected adults have a leakier blood-brain barrier than infected neonates, and adults with encephalitis have a greater viral burden in brain tissue compared to adults without encephalitis. In the choroid plexus, we discovered that despite the lack of encephalitis, neonates have an increase in monocytes and macrophages of the choroid plexus, indicating a strong immune response. While our choroid plexus epithelial cell model is still in preliminary stages, initial results are promising. Our work indicates a possible viral threshold needed for the development of encephalitis, and that the blood-brain barrier may play a role in this threshold due to lower levels of virus susceptible cells and a tighter blood brain barrier in neonates. In the choroid plexus, the strong pro- and anti-inflammatory macrophage response seen in neonates may offer an extra layer of protection development of SIVE. Our data also indicates that SIV causes a marked decrease in the expression of klotho, the anti-aging hormone that is produced in high levels in the choroid plexus in the brain. This could potentially explain the premature inflammaging phenotype seen in chronic infections. / 1 / Elizabeth Delery

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrocephalus is a disease characterized by an increase in cerebrospinal fluid (CSF) in the ventricles of the brain. This manifests as a result of either overproduction or underabsorption of CSF leading to increases in pressure, swelling and loss of brain matter. Current treatments for this disease include surgical interventions via the introduction of shunts or endoscopic third ventriculostomy, both of which aim to redirect flow of CSF in to another cavity for absorption. Limited pharmacotherapies are available in the treatment of hydrocephalus, and there exists a clinical need for drug therapies, which can ameliorate the pathophysiology associated with hydrocephalus and ventriculomegaly. CSF is produced primarily by the choroid plexus (CP), found in the ventricles of the brain. Composed of a high resistance epithelium surrounding a capillary network, the CP epithelium acts as a barrier, regulating ion transport between the CSF and blood. Transient Receptor Potential Vanilloid-4 (TRPV4) is a nonselective Ca2+-permeable cation channel expressed in the CP which is being investigated for its role in CSF production. To study hydrocephalus, we utilize two model systems; the TMEM67-/- Wpk rat, and the PCP-R cell line. The Wpk rat model is used to study the effects of drug intervention on the development and progression of hydrocephalus. The PCP-R cell line is utilized for studies which aim to understand the mechanisms by which CSF is produced. Using Ussing chamber electrophysiology, we are able to study the role of specific channels, transporters and modulators in driving epithelial ion flux across the CP. This research aims to establish a role for TRPV4 in production and regulation of CSF, and to interrogate a mechanism by which this ion transport occurs. The chapters that follow describe components of the pathway by which TRPV4 is activated and ion flux is stimulated.

The choroid plexuses are largely responsible for cerebrospinal fluid (CSF) secretion and therefore play a fundamental role in brain homeostasis. The membrane proteins involved in CSF secretion are not fully known. Several electroneutral transporters have been identified by molecular methods in choroid plexus epithelial cells but there is a lack of functional data to support their expression making it impossible to elucidate their role in CSF secretion fully. The activity of many of these transporters can be observed in cell volume regulation. Thus, the main aim of the present study was to determine the ability of mammalian choroid plexus epithelial cells to regulate their volume in response to anisosmotic challenge and to investigate the transporters involved.Experiments were performed on cells isolated from the mouse fourth ventricle choroid plexus. Cells were isolated using a combination of manual perturbation, the enzyme dispase and a Ca2+ free incubation to disrupt tight junctions. Cell volume was measured using a video-imaging method. Cells used in this study were all of a similar morphology and had a mean volume of 0.71 pL.Cells exhibited a HCO3- dependent regulatory volume increase (RVI) in response to hypertonic challenge. Strong evidence is presented that the Na+/H+ exchanger (NHE1) and the Cl-/HCO3- exchanger (AE2) contribute to the RVI but the Na+K+2Cl- cotransporter (NKCC1) and the epithelial Na+ channel (ENaC) do not. Choroid plexus cells exhibit a HCO3- dependent regulatory volume decrease (RVD) in response to hypotonic challenge. The RVD was unaffected by DIOA (an inhibitor of KCC activity), the K+ channel inhibitors TEA+, Ba2+ or 4AP or the Cl- channel inhibitors DIDS or NPPB. However removal of extracellular Ca2+ completely abolished cell swelling in response to hypotonic challenge. This sensitivity of volume change to Ca2+ was specific to cell swelling as cell shrinkage in hypertonic artificial CSF was unaffected by removal of extracellular Ca2+.Thus functional evidence is presented to further elucidate the role of several proteins in the choroid plexus cell volume regulatory response to anisosmotic challenge.

L’activateur tissulaire du plasminogène (tPA) est une protéase initialement découverte dans le sang pour son rôle fibrinolytique. C’est pour cette fonction que le tPA recombinant est utilisé pour traiter la phase aigüe de l’accident vasculaire cérébral (AVC) ischémique, même s’il présente quelques limites. Le tPA exogène peut passer du compartiment vasculaire au parenchyme cérébral où il peut influencer des processus physiologiques, et participer au devenir neuronal, notamment aggraver la mort neuronale lors d’un AVC ischémique. Le laboratoire a montré que le tPA peut traverser la barrière-hémato-encéphalique (BHE), par transcytose au travers des cellules endothéliales de la BHE et cela sous le contrôle des récepteurs LRP1 (Low density lipoprotein receptor-related protein 1). D’autres barrières existent au sein du système nerveux central notamment la barrière sang-liquide cérébrospinal (BSLCS), formée par les plexus choroïdes (PCs). Les PCs sont une route de migration pour les cellules inflammatoires et le LCS peut véhiculer des solutés, via les espaces péri-artériels, vers le parenchyme cérébral. Ainsi, dans notre première étude, nous avons testé l’hypothèse d’un passage du tPA vasculaire par les PCs. Pour cela, nous avons produit un tPA traçable in vivo et in vitro. Nous avons commencé par étudier la distribution du tPA suite à une injection intraveineuse (IV) avec comme focus les PCs et le LCS. Nos résultats montrent que le tPA exogène, suite à une injection IV, est retrouvé de manière séquentielle dans les PCs puis dans le LCS. Le tPA est donc capable de traverser les PCs. Nous avons alors développé un modèle de culture primaire de cellules épithéliales de PCs (CPECs) de souris pour disséquer le(s) mécanisme(s) sous-jacents à l’internalisation du tPA par les CPECs. Ce modèle nous a permis de montrer que l’internalisation du tPA par les CPECs est un phénomène actif, médié par un membre de la famille des récepteurs LRP, mais qui n’est ni LRP1, ni LRP2. Nous avons également mis en évidence la nécessité du domaine Finger du tPA pour son internalisation par les CPECs. Une étude préliminaire dans un modèle d’AVC suggère que l’ischémie modifie la cinétique de passage du tPA, puisqu’il y a plus de tPA dans les PCs des souris ischémiées que les souris non ischémiées.Dans une deuxième étude nous nous sommes intéressés à l’effet du tPA endogène sur les PCs. Nous montrons que l’absence de tPA endogène n’influence ni la morphologie des PCs, ni la diffusion du LCS. De plus, nous montrons que cette absence de tPA n’influence pas le nombre de macrophages et de lymphocytes T dans les PCs en conditions basales. / Tissue-type plasminogen activator (tPA) is a protease initially discovered in the blood for its fibrinolytic role. Accordingly, recombinant tPA has become the gold standard to treat the acute phase of ischemic stroke, despite some limitations. Exogenous tPA can switch from the vascular compartment to the brain parenchyma, where it can influence physiological processes, and participate in neuronal fate, including a worsening of neuronal death during ischemic stroke. In the team, it has been shown that tPA can cross the blood-brain barrier (BBB), by a transcytosis through BBB endothelial cells, under the control of LRP1 receptors (Low density lipoprotein receptor-related protein 1). The central nervous system has other barriers, including the blood-cerebrospinal fluid barrier (BCSFB), that relies on choroid plexuses (CPs). CPs are a migration route for inflammatory cells and a major source of CSF, which carries solutes to the cerebral parenchyma, via peri-arterial spaces. Thus, in a first study, we tested the hypothesis of a passage of vascular tPA through CPs. We thus produced a fluorescent tPA that can be tracked in vivo and in vitro. We first studied the distribution of tPA following intravenous (IV) injection, focusing on CPs and CSF. We show that after an IV injection, exogenous tPA is sequentially found in the CPs and then in the CSF. tPA is therefore able to cross the CPs. We then developed a model of primary culture of mouse choroid plexus epithelial cells (CPECs) to dissect the mechanism (s) underlying the internalization of tPA. This model allowed us to demonstrate that the internalization of tPA by CPECs is an active phenomenon, mediated by a member of the family of LRP receptors, but which is neither LRP1 nor LRP2. We also highlight the requirement for the Finger domain of tPA for its internalization by CPECs. A preliminary study in a murine stroke model suggests that ischemia alters the tPA passage kinetics, since there is more tPA in ischemic CPs than non-ischemic CPs.In a second study, we investigated the effect of endogenous tPA on CPs. We show that the absence of endogenous tPA influences neither CPs morphology nor CSF diffusion . Moreover we show that the absence of tPA does not influence the number of macrophages and T cells in the stroma of PCs under basal conditions.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear. Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative in vitro model is critical to elucidate the signaling pathways orchestrating CSF production in the CP. This research aims to characterize an in vitro culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrocephalus is a medical condition characterized by a buildup of cerebrospinal fluid which causes hydrostatic pressure to increase resulting neuronal destruction and can ultimately cause death. Hydrocephalus is seen in both the pediatric population and adults. Treatment of hydrocephalus usually involves surgical placement of a relocation system to drain the fluid into the abdominal cavity. Hydrocephalus may be caused by mechanical obstruction of the outflow of CSF from the ventricles or by faulty reabsorption. It can be also caused by CSF overproduction by the choroid plexus found in the lateral, third, and fourth ventricles of the brain. The choroid plexus is composed of a high resistance monolayer epithelium which surrounds a network of capillaries. Its primary function is to regulate transport of ions and water that control the production and movement of CSF. Therefore it is important to understand the mechanism of CSF production by the choroid plexus. Recently, a stable porcine choroid plexus (PCP-R) epithelial cell line with a high transepithelial resistance (TER) was developed that provides an important model to study regulation of CSF production. Ussing style electrophysiology was used to measure short circuit current (SCC) to characterize stimulated transepithelial ion transport in confluent PCP-R cells. GSK1016790, a TRPV4 agonist, was used to understand the role of TRPV4 in CSF production by the choroid plexus using PCP-R cell model. TRPV4 activation produces a sustained ion transport response that is consistent with an increase in cation secretion and/or anion absorption which is accompanied by a reversible decrease in TER. The effect of the agonist on both SCC and TER was blocked by HC067047, a TRPV4 antagonist, showing that the sustained ion transport and TER change is TRPV4 specific. TRPV4 mediated ion flux was inhibited by CFTR inhibitor II GlyH-101, a cell permeable inhibitor of the cAMP activated chloride channel CFTR, when added on either side of the membrane and was not accompanied by a TER reversal which showed that CFTR is activated by TRPV4 mediated ion flux. TMEM16A, a calcium activated chloride channel, was speculated to be located in that basal membrane as T16Ainh-AO1, a membrane permeable TMEM16A inhibitor, reversed the TRPV4 mediated ion flux when added on either side of the membrane. Slight reversal in TER was observed when T16Ainh-AO1 was added on the apical side. Apamin, a differential inhibitor of calcium activated small conductance potassium channel 1, 2 and 3 (SK1, SK2 and SK3) had no effect on the TRPV4 mediated ion flux. Whereas, fluoxetine, a membrane permeable inhibitor of SK1, SK2 and SK3 channel, inhibited the TRPV4 mediated ion flux and TER change. Bumetanide, an inhibitor of the sodium-potassium-chloride cotransporter reversed TRPV4 mediated ion flux when added on the apical membrane but not on the basal membrane indicating a possible K+ secretion via SK1 and/or SK4/IK channels and Cl- absorption through CFTR and TMEM16A channels. Acetazolamide, a carbonic anhydrase inhibitor and a compound used to treat hydrocephalus had no effect on the TRPV4 mediated ion flux. cAMP is an intracellular mediator involved in neuromodulator effects, inflammatory responses and other regulatory mechanisms and is constitutively activated by forskolin. In PCP-R cells, forskolin stimulated an increase in transepithelial ion flux that is consistent with an increase in cation absorption and/or anion secretion. Forskolin mediated ion transport was inhibited by CFTR inhibitor II GlyH-101 when added on either side of the membrane. No change in TER was observed. No effect on forskolin mediated ion flux was observed when T16Ainh-A01, apamin or fluoxetine were added. Forskolin stimulated transport is partially inhibited by 1 mM BaCl2. Barium chloride is a general inhibitor of K+ channels. No change in TER was observed.

<p>  </p> <p>The choroid plexus (CP) in brain ventricles secrete cerebrospinal fluid (CSF) that bathes the adjacent subventricular zone (SVZ); the latter is the largest neurogenic region in adult brain harboring neural stem/progenitor cells (NSPCs) and supplies newborn neurons to the olfactory bulb (OB) for normal olfaction. We discovered the presence of a CP-SVZ regulatory (CSR) axis in which the CP, by secreting small extracellular vesicles (sEVs), regulated adult neurogenesis in the SVZ and maintained olfaction. The proposed CSR axis was supported by 1) differential neurogenesis outcomes in the OB when animals treated with intracerebroventricular (ICV) infusion of sEVs collected from the CP of normal or manganese (Mn)-poisoned mice, 2) progressively diminished SVZ adult neurogenesis in mice following CP-targeted knockdown of SMPD3 to suppress CP sEV secretion, and 3) compromised olfactory performance in these CP-SMPD3-knockdown mice. Collectively, our findings demonstrate the biological and physiological presence of this sEV-dependent CSR axis in adult brains.</p>

Acetazolamide (AZA), the only drug approved for treatment of hydrocephalus, is effective in only 25-30% of patients while its effect on fluid flow in the choroid plexus (CP) is unknown. The drug reversibly inhibits Aquaporin 4 (AQP4), the most highly expressed „water pore‟ in the brain, and it is postulated that it reduces cerebrospinal fluid (CSF) production by modulating AQP1 (mostly found in the apical membrane of the CP). In this study, we sought to elucidate the effect of AZA on AQP1 and fluid flow in CP. Primary CP culture from p10 Sprague-Dawley rats and TRCSF-B cell line were grown on Transwell permeable supports, treated with 100µM AZA or 100µM Vinpocetine (previously shown to increase AQP1 levels), and tested by: a) Fluid assays using TRITC-labeled Dextran to assay direction and extent of fluid flow; b) Immunoblot, Immunocytochemistry (ICC), and RT-PCR for AQP1 expression. Immnoblots and ICC analyses showed that AQP1 protein levels decrease in a delayed manner (lowest at 12 hours) with AZA treatment. The reduction in AQP1 protein was transient and preceded by a reduction in mRNA levels (lowest at 6 hours). Transwell fluid assays indicate a shift in fluid flow at 2 hours, prior to the changes in AQP1 mRNA or protein. Alteration of fluid flow by AZA (in both primary culture and TR-CSFB) is similar to Vinpocetine‟s effect in primary culture. Together with druginduced alterations in AQP1 levels, these data suggest independent mechanisms behind fluid flow and AQP1 expression.

Indiana University-Purdue University Indianapolis (IUPUI) / Pediatric hydrocephalus is a complex neurological condition associated with a pathological accumulation of cerebrospinal fluid (CSF), typically within the brain ventricular system. Pediatric hydrocephalus can be primary (due to genetic abnormalities or idiopathic causes), or secondary to injuries such as hemorrhage, trauma, or infection. The current permanent treatment paradigms for pediatric hydrocephalus are exclusively surgical and include the diversion of CSF via shunt or ventriculostomy. These surgical interventions are wrought with failures, burdening both the United States healthcare system and patients with repeat neurosurgical procedures. Thus, the development of nonsurgical interventions to treat hydrocephalus represents a clinically unmet need. To study hydrocephalus, we use a genetic rat model of primary neonatal hydrocephalus, the Tmem67P394L mutant. In several proof-of-concept studies, we identify antagonism of the transient receptor potential vanilloid 4 (TRPV4) channel and associated upstream regulatory kinase, serum-andglucocorticoid-induced kinase 1 (SGK1) as therapeutics for the treatment of hydrocephalus. Using in vitro models of the choroid plexus epithelium, the tissue which produces CSF, we show compelling proof-of-mechanism for TRPV4 antagonism and SGK1 inhibition at preventing CSF production. Therefore, the studies in this dissertation provide substantive evidence on the role of TRPV4 in the choroid plexus in health and disease.

The choroid plexus is an organ within each brain ventricle comprised of elaborate folds of epithelium (CPe) and vasculature. It performs numerous functions essential for brain development and health, including secretion of cerebrospinal fluid (CSF) and acting as the blood-CSF barrier. Functionality requires: (1) that CPe and vasculature develop in register and in close proximity, so that the CPe ensheaths the vasculature at a high surface area to volume ratio, which permits efficient CSF secretion; and (2) that CPe barrier integrity is sustained throughout choroid plexus expansion. Genetic experiments in mouse embryos have identified a central role for Sonic hedgehog (Shh) in coordinating these developmental challenges. Specifically, Shh is secreted by differentiated CPe and drives choroid plexus expansion. In the absence of Shh, a hypoplastic choroid plexus forms, which is deficient in CPe, vasculature, and villous folds. Two choroid plexus cell populations respond to Shh: (1) rhombic lip-resident CPe progenitor cells and (2) vascular pericytes. Here, I present evidence that canonical Shh signaling to CPe progenitors alone is insufficient to fully drive their proliferation at normal rates. Rather, Shh-triggered pericytes appear to secondarily boost CPe progenitor cell proliferation, in addition to acting in vascular development. Shh-triggered pericytes also appear necessary for formation of the characteristic folds of the choroid plexus. Thus, pericytes coordinate the expansion of choroid plexus epithelium and vasculature. Notch signaling was also explored and was found to inhibit the differentiation of CPe progenitors, maintaining them in a proliferative state. Notch activation in CPe progenitors leads to invaginated tubules from the overproliferating CPe progenitor domain, without associated vascular growth or villous folds. Folding morphogenesis may thus be regulated by vascular components such as pericytes, and require that vascular growth match CPe growth. To identify Shh-induced pericyte signaling programs that might underlie these developmental processes, expression profiling was performed on dsRed-labeled pericytes isolated from Shh-deficient versus wild-type choroid plexuses. Candidate genes, including several involved in lipid metabolism, were identified. Collectively, this work points to pericytes as central in orchestrating the coordinated elaboration of multiple choroid plexus cell types, producing the complex tissue architecture required for efficient CSF production.