Browsing by Subject "TRPV4"

Now showing 1 - 6 of 6
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    Investigating TRPV4 Signaling in Choroid Plexus Culture Models
    (2022-05) Hulme, Louise; Blazer-Yost, Bonnie; Baucum, AJ; Mastracci, Teresa; Belecky-Adams, Teri
    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.
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    Porcine choroid plexus-Riems cell line demonstrates altered polarization of transport proteins compared with the native epithelium
    (American Physiological Society, 2022) Hochstetler, Alexandra; Hulme, Louise; Delpire, Eric; Schwerk, Christian; Schroten, Horst; Preston, Daniel; Simpson, Stefanie; Blazer-Yost, Bonnie L.; Biology, School of Science
    The choroid plexus epithelium (CPe) forms a barrier between the cerebral blood supply and the cerebrospinal fluid (CSF), establishing the blood-CSF barrier (BCSFB). CSF is actively secreted by the CPe via tightly controlled processes involving multiple channels, transporters, and pumps. The importance of controlling CSF production and composition has been accentuated recently with an appreciation of CSF dysfunction in many pathologies. For mechanistic studies of CSF production, isolated CPe cell lines are valuable for the testing of hypotheses and potential drug targets. Although several continuous CPe cell lines have been described, none appear to have all the characteristics of the native epithelium and each must be used judiciously. The porcine choroid plexus-Riems (PCP-R) cell line forms a high-resistance monolayer characteristic of a barrier epithelium. Conservation of this phenotype is unusual among CPe cell lines, making this model useful for studies of the effects of infection, injury, and drugs on permeability. We have recently discovered that, although this line expresses many of the transporters expressed in the native tissue, some are mispolarized. As a result, inferences regarding fluid/electrolyte flux and the resultant CSF production should be pursued with caution. Furthermore, extended culture periods and changes in media composition result in significant morphological and functional variability. These studies provide a more detailed characterization of the PCP-R cell line concerning transporter expression, polarization, and functionality, as well as plasticity in culture, with the goal to provide the scientific community with information necessary to optimize future experiments with this model.
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    Role of Choroid Plexus TRPV4 Channel in Health and Disease
    (2022-08) Hochstetler, Alexandra; Blazer-Yost, Bonnie L; Berbari, Nicolas; Baucum II, AJ; Roper, Randall; Raskin, Jeffrey
    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.
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    TRPV4 and cAMP Mediated Ion Transport in the Porcine Choroid Plexus
    (2016-12-01) Ahmed, Shehab; Blazer-Yost, Bonnie
    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.
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    TRPV4 Implications in Inflammation and Hydrocephalic Neurological Disease
    (2019-05) Simpson, Stafanie J.; Blazer-Yost, Bonnie; Belecky-Adams, Teri; Berbari, Nicolas; Goodlett, Charles
    Hydrocephalus is a debilitating disease characterized by an increase in cerebrospinal fluid (CSF) in the brain, leading to increases in pressure that can ultimately result in death. Current treatments for hydrocephalus include only invasive brain surgery. Therefore, the need for a pharmaceutical therapy is great. In order to develop a suitable treatment, we first must be able to study the disease and the mechanisms by which it develops. By characterizing appropriate in vivo and in vitro models, we are better able to study this disease. In this thesis, the Wpk rat model and the PCP-R cell line are described as such appropriate models. In addition to suitable models, we also require a target for drug treatment. Transient Receptor Potential Vanilloid 4 (TRPV4) is a non-selective cation ion channel present in the main CSF-producing organ in the brain, the choroid plexus (CP). Preliminary data suggest this channel plays a role in the development of hydrocephalus. In the following work, some of the mechanisms by which TRPV4 functions in the brain are also described, including through calcium-sensitive potassium channels and inflammation. From this research, we are able to achieve a better understanding of the function of TRPV4 and how it can affect the development and progression of hydrocephalus.
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    TRPV4 in the Choroid Plexus Epithelium: Pathway Analysis and Implications for Cerebrospinal Fluid Production
    (2019-12) Preston, Daniel; Blazer-Yost, Bonnie; Belecky-Adams, Teri; Clack, James; Berbari, Nick
    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.